Tumor suppressor gene CDKN2 (also called MTS1, CDK4I and p16INK4) is located in 9p21 and deleted homozygously in a high percentage of tumor cell lines. We have examined the sequence of CDKN2 in 154 tumor cell lines that are not homozygously deleted for CDKN2. Overall, 18% (27/154) of the cell lines carried mutations in CDKN2. These mutations were found in cell lines derived from melanoma, bladder, lung and prostate cancers, as well as sarcomas of various origin. The spectrum of the CDKN2 mutations found in melanoma cell lines indicated a major role for ultraviolet light in generating the mutations, suggesting the mutations occurred in vivo. The frequency of loss of heterozygosity in 9p21 in this set of lines is only slightly higher than the background rate of aneuploidy, suggesting that a second 9p21 tumor suppressor gene, if it exists, must lie near CDKN2.