Enterococci, although part of the normal ffora of the human gastrointestinal tract, have been recognized as an important cause of nosocomial infection for over two decades (8, 11) and are commonly implicated in urinary tract infections, bacteremia, intra-abdominal and surgical wound infections, catheterrelated infections, and endocarditis (11). Enterococci have also been associated with the production of biofilms in root canals (3), on different biomaterials (1, 7, 9, 20), and on various indwelling medical devices, such as ureteral stents (6), intravascular catheters (17), and silicone gastrostomy devices (2). The genetic determinants involved in biofilm formation by Enterococcus faecalis have just begun to be understood (4, 5, 7, 9, 13, 19, 20). Several studies have evaluated esp (9, 19, 20), including one recent study which reported that the presence of esp was significantly associated with the ability to form biofilm on abiotic surfaces (20); that study also found that insertional inactivation of esp of two strains, but not of another, markedly impaired biofilm production. We have reported a lack of association between esp presence and the presence or absence of biofilm among a large collection of clinical isolates from various sources; however, we found that greater amounts of biofilm formation were highly significantly associated with the presence of esp (9). We also found that endocarditis isolates more often made biofilm than nonendocarditis isolates and also made significantly more biofilm (9). We further demonstrated that disruption of epa (enterococcal polysaccharide antigen), atn (autolysin), the fsr locus (E. faecalis regulator), and gelE (gelatinase) genes of E. faecalis OG1RF (an esp-negative strain) markedly reduced biofilm formation in a microtiter plate assay as well as by phase-contrast microscopy (9); this is consistent with other reports about the importance of gelatinase for E. faecalis and the production of biofilm (4, 7, 9, 10, 13). The marked impact of gelE disruption on biofilm production led us to speculate that biofilm production was likely to be greater among naturally occurring gelatinase-producing isolates than among non-gelatinase-producing isolates. To test this hypothesis, we examined the 163 isolates previously analyzed for esp and biofilm (using a microtiter plate assay after 24 h of growth and crystal violet staining [9]) for the production of gelatinase (15, 16) and for the presence of gelE and fsr genes, and we correlated these results with biofilm production. Among the 163 isolates tested, 71 were GelE and 92 (56%) were GelE (all 92 were fsrB gelE)(Table 1)(16). Eightyfour of the 92 GelE isolates were biofilm producers (optical density at 570 nm [OD570] 0.5), while 67 of the 71 GelE isolates produced biofilm (P 0.55, Fisher’s exact test); the median biofilm ODs for GelE and GelE isolates were also essentially equal (1.457 versus 1.488)(P 0.43, Mann-Whitney test). For the subgroup of esp-lacking isolates, the median biofilm OD of GelE isolates was higher, although not significantly so, than that of GelE isolates (median OD, 1.155 for the 19 esp-lacking/GelE isolates versus 1.424 for the 70 esp-lacking/GelE isolates), suggesting that GelE may contribute to biofilm in the esp-lacking background. It is of interest (Table 1) that the majority of isolates (122) had either only gelatinase production or only esp, with fewer strains having (22) or lacking (19) both. We also noted that the biofilm ODs were highest for the 22 esp/GelE isolates (median OD, 1.633; interquartile range [IQR], 1.178 to 2.456) and lowest for the 19 esp-lacking/GelE isolates (median OD, 1.155; IQR, 0.586 to 1.746)(P 0.02, Mann-Whitney test), consistent with our previous observation that esp …