The p16 gene has been identified as a candidate tumour suppressor gene at 9p21, a region commonly deleted in bladder cancer. We screened 140 bladder tumours and 16 cell lines for deletions and sequence variants of p16. Eight cell lines showed homozygous deletion of p16 and two had small sequence variations. All 13 tumours with small defined deletions of 9p21, 18/31 (58%) of tumours with monosomy 9 and 9/91 (10%) of tumours with no chromosome 9 loss of heterozygosity had homozygous deletion of p16. No tumour-specific sequence variants were identified. Deletion mapping revealed a nested set of deletions focused on p16. Six deletions involved p16 but not the related and adjacent gene p15 and one tumour had an intragenic deletion of p16. All other deletions involved both p16 and p15. We conclude that p16 represents the major target for deletion at 9p21 in bladder cancer.