Deletions of the long arm of chromosome 9 are the most common genetic alteration in transitional cell carcinoma (TCC) of the bladder. Several regions of deletion on 9q have been mapped by loss of heterozygosity (LOH) analysis, one of which encompasses one of the two loci for tuberous sclerosis, TSC1, at 9q34. Tuberous sclerosis complex (TSC) is an autosomal dominant condition in which affected individuals develop benign tumors (hamartomas) in many organs. There is a small increase in risk of renal cell carcinoma (<2%), but the hamartomas are of stromal origin and patients do not develop an excess of epithelial malignancies. However, during a search for candidate bladder tumor suppressor genes within the 9q34 region of LOH, we previously found a small number of mutations of TSC1, raising the possibility that this represents a bladder tumor suppressor. Here, we have carried out mutation analysis of 62 bladder tumors and 33 bladder tumor-derived cell lines to establish the frequency and spectrum of TSC1 mutations in TCC. Twelve percent of samples contained mutations. We found 10 somatic mutations, 9 of which are novel mutations not found previously in TSC cases. Two of these were missense mutations, a type of change only rarely observed in the germ line in TSC. We also identified a bladder tumor patient carrying a germ-line mutation but with no symptoms of TSC. The tumor in this case and in two other cases with somatic mutations retained the wild-type allele. Thus 3 cases with mutation retained heterozygosity for TSC1 despite our selection of tumors mostly with 9q LOH (>80%) for the study. This may indicate that haploinsufficiency for TSC1 can contribute to the development of bladder cancer and, if so, that the LOH of TSC1 observed in >50% of TCCs is biologically significant.