Alterations of the CDKN2A locus on chromosome 9p21 encoding the p16INK4A cell cycle regulator and the p14ARF1 p53 activator proteins are frequently found in bladder cancer. Here, we present an analysis of 86 transitional cell carcinomas (TCC) to elucidate the mechanisms responsible for inactivation of this locus. Multiplex quantitative PCR analysis for five microsatellites around the locus showed that 34 tumors (39%) had loss of heterozygosity (LOH) generally encompassing the entire region. Of these, 17 tumors (20%) carried homozygous deletions of at least one CDKN2A exon and of flanking microsatellites, as detected by quantitative PCR. Analysis by restriction enzyme PCR and methylation-specific PCR showed that only three specimens, each with LOH across 9p21, had bona fide hypermethylation of the CDKN2A exon 1alpha CpG-island in the remaining allele. Like most other specimens, these three specimens displayed substantial genome-wide hypomethylation of DNA as reflected in the methylation status of LINE L1 sequences. The extent of DNA hypomethylation was significantly more pronounced in TCC with LOH and/or homozygous deletions at 9p21 than in those without (26% and 28%, respectively, on average, versus 11%, p < 0.0015). No association of LOH or homozygous deletions at 9p21 with tumor stage or grade was found. The data indicate that DNA hypermethylation may be rare in TCC and that deletions are the most important mechanism for inactivation of the CDKN2A locus. The predominance of allelic loss may be explained by its correlation with genome-wide DNA hypomethylation, which is thought to favor chromosomal instability and illegitimate recombination.