Studies of second, non-ocular tumours in surviving retinoblastoma patients and their families have reported a higher than expected incidence and lower age at diagnosis of bladder tumours. This suggests that RB mutations may predispose to bladder cancer. To determine whether this gene is involved in the development of sporadic bladder tumours we have examined 162 bladder tumours for evidence of structural alterations to the RB gene. Ninety-four patients were informative with one or more intragenic RB probes, and 28 of these (29%) showed loss of heterozygosity (LOH). Of these, two tumours showed homozygous deletions with the 5' intragenic probe p123M1.8. The probe p68RS2.0, which recognizes a variable number of tandem repeats site in intron 17 of the RB gene, detected new alleles in 5 of 162 tumours, one of which also showed LOH at another polymorphic site within the gene. The 28 tumours with RB LOH were screened with the RB cDNA probes pR3.8 and pR0.9, which revealed two homozygous deletions and one rearrangement. The tumours with RB LOH were also screened for loss of three markers which flank RB, D13S1 which maps proximal and D13S2 and D13S3 which map distal to RB on chromosome 13q. Two tumours showed retention of heterozygosity for flanking markers on one side of RB and another for markers on both sides. These results suggest that RB is the target gene on 13q in these bladder tumours. When RB loss was compared with tumour grade and stage, an association between high tumour grade and RB loss (0.005 greater than P greater than 0.001) and between muscle invasion and RB loss (P greater than 0.001) was found. Twenty-six of the 28 tumours with LOH were muscle-invasive. This represents 56% of invasive tumours. Only 2/48 (4%) superficial tumours showed RB allele loss, and one of these has progressed rapidly to invasive disease. These results show that LOH at the RB locus is a frequent genetic event in bladder tumours and may identify a subset of more aggressive tumours.