Multiple tumor suppressor gene 1 (MTS1) has been found mutated or deleted in a variety of human cancers. Our purpose was to identify and characterize MTS1 gene mutations in primary oral squamous cell carcinomas (SCCs) in each of the three exons of the MTS1 gene. Seventeen archival samples of oral SCC were evaluated for the presence of MTS1 mutations using single strand conformation polymorphism (SSCP) and DNA sequencing. Three of 17 tumors exhibited MTS1 gene mutations: one tumor exhibited a mutation in exon 2 and two tumors exhibited mutations at the splice site junction of intron 2 and exon 3. Three tumors also exhibited a common base change in the 3' untranslated region of exon 3, which is interpreted as a likely polymorphic variant. An examination of the three tumors exhibiting MTS1 point mutations revealed no unique characteristics relative to p53 immunohistochemical activity, mitotic frequency, or degree of histologic differentiation. This study indicates that MTS1 gene mutations may be involved in at least a minor proportion of oral SCCs.