We have examined the chicken cytoskeletal actin gene for promoter activity and for the presence of cis-acting gene transcription activating sequences. Plasmids were constructed with the β-actin promoter or other fragments of the β-actin gene adjacent to either the truncated Herpes simplex virus (HSK)-tk promoter driving the neo gene or the enhancerless simian virus 40 (SV40) promoter driving the chloramphenicol acetyltransferase (CAT) gene. The neo plasmids were tested for frequency of transformation of mammalian cells to G418 resistance. The CAT constructions were tested for CAT expression in both transient and stable expression systems. We find that the β-actin promoter is very strong in all of the assay systems and is as strong as any promoter we have tested. Also, we find that there are sequences in the vicinity of the β-actin promoter which act like an enhancer sequence in activating transcription from the truncated HSV-tk promoter and the enhancerless SV40 promoter. Constructs with these actin sequences augment the transformation frequency of the neo plasmids, and stimulate the level of CAT expression from the CAT plasmids after stable chromosome insertion but not during the transient expression phase