Homozygous and hemizygous deletions of 9p21 are the earliest and most common genetic alteration in bladder cancer. The identification of two cell cycle regulators, CDKN2 and CDKN2B, that map to the common region of deletion has prompted the hypothesis that they are critical tumor suppressor genes in this malignancy. However, controversy as to whether these genes are the only or even the most important target in bladder cancer oncogenesis remains. To more clearly determine the effect of these 9p21 alterations, we mapped the homozygous deletions and performed a detailed mutational and expression analysis for CDKN2, CDKN2B and a closely linked gene, methylthioadenoside phosphorylase (MTAP), in 16 established bladder cancer cell lines. Nine of the 16 lines exhibit large (30 to > 2000 kb) homozygous deletions on 9p21. All deletions include at least one exon of CDKN2, eight of nine include CDKN2B, and six of nine include MTAP. MTAP function correlates with the genomic deletions. SSCP and sequence analysis does not reveal any inactivating point mutations of CDKN2 or of CDKN2B in any of the cell lines without homozygous deletions, and all express the CDKN2 and the CDKN2B mRNA as well as the encoded p16 protein. The p16 protein levels vary widely and are correlated with absent pRb expression. We conclude that the 9p21 deletions in bladder cancer usually inactivate the CDKN2. CDKN2B, and MTAP genes but that CDKN2 is the most common target. Other mechanisms for inactivating this gene in bladder cancer appear to be uncommon.