Caveolin-1, a tumor suppressor gene, was found to be inactivated by structural abnormalities or epigenetic changes in several types of human cancers. In order to investigate the role of Caveolin-1 in the pathogenesis of human oral squamous cell carcinoma, the exon 1 and 3 sequences of the Caveolin-1 were investigated in 74 oral squamous cell carcinomas and 15 oral cancer cell lines. In addition, Caveolin-1 expression was examined in 15 oral cancer cell lines. Mutations of the Caveolin-1 gene were identified in five cancers (1 missense and 4 silent mutations), with all mutations being detected in exon 3. Among the cancers containing the Caveolin-1 mutation, case 53 harbored a missense mutation from Ile (ATT) to Phe (TTT) at codon 141 and two silent mutations at codons 112 and 163 of the Caveolin-1 gene. Cases 27, 44, and 84 had two silent mutations from GAC (Asp) to GAT (Asp) at codon 82, and from CCA (Pro) to CCT (Pro) at codon 132. Case 89 contained three silent mutations at codons 84, 124, and 161. However, mutations of the Caveolin-1 gene were not observed in normal human oral keratinocytes, the human papillomavirus-immortalized oral keratinocytes, and 15 oral cancer cell lines. Despite the intact Caveolin-1 gene, one oral cancer cell line, HEp-2, lacked the caveolin-1 protein. In contrast, two cancer cell lines, KOSCC-25C and -33B, had an insignificant amount of the caveolin-1 protein. These results provide genetic evidence showing that the inactivation of Caveolin-1 by a mutation or by reduced expression may play a role in the pathogenesis of oral cancer.