Mutation of the p53 tumor suppressor gene has been demonstrated in a large proportion of human head and neck squamous cell carcinomas (HNSCCs) and has been assumed to play a role in the pathogenesis of these tumors, although no formal evidence of functional aberration has been demonstrated. In this study, we isolated cDNA clones encoding the entire p53 coding region from six human HNSCC cell lines that showed aberrant patterns of p53 expression in the parental cells, analyzed their nucleotide sequences, and characterized their function in vivo. cDNAs cloned from four cell lines harbored alterations within the p53 coding sequence (one missense mutation, one missense mutation plus in-frame deletion, one splice donor mutation, and a 1-nt insertion). HN30 cells, which contained wild-type p53 nucleotide sequences, showed a high constitutive level of protein expression. HN26 cells contained wild-type coding sequences but did not express the 53-kDa protein, although the mRNA was transcribed and a molecule of increased molecular mass (70 kDa) was observed by western blotting. Functional studies revealed that none of the four proteins encoded by mutant cDNAs were able to transactivate expression of a reporter plasmid containing a wild-type p53 consensus binding site when cotransfected into p53-null cells, whereas molecules encoded by wild-type p53 cDNAs increased reporter gene expression about a hundredfold over uninduced levels. Co-expression of each mutant cDNA with wild-type p53 cDNA and a wild-type p53-responsive reporter gene demonstrated that each of the proteins encoded by mutant cDNAs harbored some degree of inhibitory activity that varied depending on the mutation present. Thus, aberrant p53 function as a result of mutation or altered expression characterizes oral squamous cell carcinomas. The inhibitory activity of these molecules may be a mechanism for deregulation of the function of co-expressed wild-type p53 that may be of importance during the early stages of tumor development.