In an attempt to examine whether the inactivation of p16 INK4a is an important early event in the development of sporadic melanoma in vivo, we have systematically analysed 46 uncultured primary cutaneous melanomas. Loss of heterozygosity (LOH) of chromosome region 9p21-22 (where the p16 INK4a resides) was detected in 11 tumours (24%) by PCR-based LOH analyses. Direct sequencing of all three exons of the p16 INK4a gene in these 11 tumours revealed no somatic mutation although germline mutations which have not been reported previously as common polymorphisms were detected in two patients. Further sequencing analyses of the p16 INK4a gene exon 2 in 19 additional tumours with no evidence of LOH on 9p21-22 identified only one heterozygous C–> T mutation at codon 81 altering a proline to a leucine. A sensitive methylation-specific PCR assay did not reveal de novo methylation of the 5′ CpG island in exon 1 of the p16 INK4a gene in any of the tumours showing 9p21-22 allelic loss or a heterozygous p16 INK4a mutation. Complete loss of p16 INK4a protein, most likely due to homozygous deletion of the p16 INK4a gene, was observed in 6 (15%) out of 39 evaluable cases by immunohistochemical analyses on frozen sections using two different anti-p16 INK4a antibodies. The results show that inactivation of p16 INK4a is not as frequent in primary melanoma as has been reported in cell lines, and warrant further search for another tumour suppressor on 9p21-22. This study also emphasizes the importance of examining uncultured primary tumours rather than cell lines to define early events in tumorigenesis.