The INK4A locus on 9p21 is deleted or rearranged in a large number of human cancers. The locus encodes two unrelated and independently acting negative cell-cycle regulators, p16 and p19ARF, arising in alternate reading frames from a partly shared sequence. We analyzed five human melanoma cell lines for deletions at the INK4 loci and flanking microsatellite markers on 9p21. All the cell lines displayed deletions of varying sizes. The metastatic cell line IGR-1 showed a large deletion between the markers D9S736 and D9S171. In the cell lines WM-115 and WM-266-4, the deletion included exon 1alpha of p16, exon 1beta of p19ARF, and exon 2 of the INK4B (p15) gene. Two cell lines, SK-MEL-5 and A2058, had deletions confined to exon 1beta and the microsatellite marker D9S942. RT-PCR experiments showed the presence of the p16 and p15 transcripts and absence of p19ARF expression in both SK-MEL-5 and A2058 cell lines. The selective loss of the exon 1beta of p19ARF and retention of the p16 and p15 genes and their expressions in these two cell lines support the putative tumor suppressor role for the alternate reading frame p19ARF gene.