Mutations in the p53 tumor suppressor gene have been found to be the most frequent genetic alterations in human malignancies. To further examine the idea that neoplastic progression is associated with mutations in the p53 gene, we analyzed matched primary and metastatic tumor samples. The samples included 15 pairs of breast cancer and metastases to lymph nodes, four pairs of gastrointestinal adenocarcinomas and metastases to liver, one colon adenocarcinoma and metastasis to a lymph node, and one lung carcinoma and metastasis in the pleura. Genomic DNA or cDNA from each tumor sample was amplified by the polymerase chain reaction and labeled by using one biotinylated primer. The DNA strands were separated with magnetic streptavidin beads and sequenced directly. p53 mutations were detected in 11 of 21 patients (52%) in either primary tumors, metastases, or both. In six of these patients the primary tumor and matched metastasis shared the same single mutation. In the other patients an additional mutation in the primary tumor only or a mutation in the metastasis only was observed. Our data suggest that tumor development and progression toward metastasis involves structural alterations in the p53 gene that occur early in carcinogenesis. In some cases, genetic changes in metastatic spreading may also include the appearance of a mutation in a metastasis derived from a primary tumor expressing wild-type p53, a selection of metastatic cells with a single mutation from a primary tumor expressing two different mutations, or loss of heterozygosity.