Homozygous deletions of 9p21, including the cyclin-dependent kinase inhibitor genes p16INK4 and p15INK4B, have been reported frequently in melanoma (as well as other tumor) cell lines. Germline mutations within the p16INK4 gene have also been described in a proportion of familial melanoma kindreds, suggesting that p16INK4 is the 9p21 "melanoma" gene. We have previously concluded that deletion of this chromosomal region can occur early (before metastasis) and in vivo in sporadic melanoma due to the identification of identical hemizygous losses on 9p21 in six autologous melanoma cell lines established from an individual patient (DX). These related cell lines have now been used to evaluate the timing of deletion/mutation of the p16INK4 and p15INK4B genes during tumor progression in melanoma. Surprisingly, homozygous deletions of a < or = 200-kb region surrounding p15INK4B, but not p16INK4, were detected in all six cell lines. Furthermore, single strand conformation polymorphism and sequencing analysis of the remaining p16INK4 allele in each case revealed only one intragenic mutation (in DX-6), whereas Western analysis provided evidence that p16INK4 protein was expressed in all six instances. These findings, taken together with those generated on other unrelated melanoma tumors and cell lines, suggest that hemizygous loss (or haplo-insufficiency) of the p16INK4 gene may be enough to place a melanocyte on a tumor pathway, and/or that the p16INK4 gene is not the sole 9p21 locus targeted in sporadic melanoma.