Mutations in the human homologue of Drosophila patched (PTCH) have been identified in patients with nevoid basal cell carcinoma syndrome (NBCCS; also called Gorlin syndrome) as well as sporadic basal cell carcinomas and medulloblastomas. However, using PCR-SSCP analysis, mutations in PTCH have been found in only a fraction (about one third to a half) of NBCCS patients. In this study, we determined the whole genomic organizations of the PTCHgene and developed a new set of more accurate primers for the analysis of mutations in PTCH. Using these primers, we examined 8 Japanese NBCCS patients for mutations in all PTCH exons by direct sequencing of the PCR products. As a result, we identified 5 novel PTCH mutations in 6 out of 8 patients including 2 sisters as well as 5 polymorphisms, two of them, 1704G>C and 2928G>C were novel. Four of these mutations, 900delC, 1247insT, 1999delC and 933+5G>T, cause protein truncation due to the insertion or deletion of a single nucleotide or aberrant splicing. The remaining mutation, 1514G>A was a missense alteration (G509D). Interestingly, the amino acid substitution, G509V, has been reported previously in an NBCCS patient, suggesting an important role of this amino acid residue in the function of PTCH protein. The difference in the detection rate of PTCH mutations among NBCCS between previous reports and ours is due to the difference either in ethnicity or in the detection methods. © 2003 Wiley-Liss, Inc.