Data on p53 alterations in human cancers are mainly based on studies restricted to the core domain (exons 5-9), because mutations outside this region are assumed to be rare. To test this assumption, we studied 25 consecutive patients with primary, untreated head and neck squamous cell carcinoma (HNSCC) with a p53 mutation analysis strategy that consists of sequencing all 11 p53 exons and the complete p53 mRNA. With this method, we encountered p53 mutations in 91% of patients; 33% of these were located outside the core domain. Overexpression of the p53 protein was assessed with staining with antibody Bp 53-12-1. Protein overexpression was found in 64%. In one case, p53 overexpression occurred without p53 gene mutations. Analysis of tumor tissue from two autopsied patients with multiple lesions in the head and neck and at distant sites allowed analysis of the clonal relationship of the different tumor foci. In one patient, the head and neck lesion had a mutation different from the one observed at the distant sites, suggesting two different primary tumors, one of them leading to widespread metastastic disease. In all lesions from the second patient, the same mutation was found, suggesting one primary that had metastatized. It appears that sequencing of all exons of the p53 gene is vital for assessment of the real incidence of p53 mutations in HNSCC, because 33% of all mutations are located outside the core domain, leading to a mutation frequency of almost 100% in HNSCC. In 96% of cases, either presence or absence of p53 protein expression could be explained by the type of p53 gene mutation. When only analyzing the p53 core domain, the incidence of p53 mutations in HNSCC is underestimated.