LOH analysis suggests that multiple tumor suppressor genes play a role in the development of human TCC. The human homolog of the Drosophila PTCH was recently cloned and mapped to the BCNS locus on 9q22.3, a chromosomal region commonly deleted in TCCs. We first examined the steady state mRNA transcription of the PTCH, SMOH and GLI3 genes of the HH signal transduction pathway in TCC cell lines and normal urothelium. Normal urothelium and TCC cell lines express these three genes within the PTCH signal transduction pathway. We then screened for PTCH mutations in 'hot spot' exons 6, 8, 13 and 16 by PCR/SSCP analysis of genomic DNAs from 54 TCC tumor samples and control autologous peripheral blood lymphocytes. DNA sequence analysis confirmed TCC-specific mutations in two of 54 patients (3.7%). These mutations resulted a single amino acid substitution and two frame shifts. One tumor had PTCH mutations in exon 16 as well as exon 13 and one tumor had a mutation in exon 13 alone. Both TCC tumors that contained PTCH mutations had a loss of heterozygosity at 9q. Although the PTCH protein has an unknown function in urothelial cells, the detection of the PTCH, SMOH and GLI3 transcripts in normal urothelium and TCC cell lines and rare PTCH mutations in tumor samples suggest that the HH pathway may have a role in controlling the proliferation of urothelial cells and that PTCH mutations may contribute to the development of a subset of TCCs.