We have looked for LOH, mutation, and expression of p53 in a series of 56 primary gastric carcinomas, using Southern blotting, constant denaturant gel electrophoresis (CDGE), direct sequencing, and immunohistochemistry. Two hundred-four lymph node metastases from 36 of these cases were also studied by immunohistochemistry. Loss of constitutional heterozygosity for the p53 locus was detected in five of 16 informative cases; in four of these cases there was a concurrent point mutation at the retained allele. Immunoreactivity for p53 product and mutation of p53 were observed in nine and 10 of the 56 tumors, respectively. There was no significant relationship between p53 immunoreactivity or mutation and pathologic features. The overall prevalence of p53 mutations, as detected by CDGE, was 18%. Direct sequencing confirmed p53 mutations (all G:C-A:T transitions) in seven cases, six of which were missense mutations; the remaining case was a silent mutation. In three of the cases with missense mutations, these occurred at CpG dinucleotides (codons 175, 273, and 282). Three cases with immunoreactivity for p53 product in the primary tumor also showed positive staining in the metastases. In two of these cases (with six and nine metastases, respectively) a positive staining was observed in all lymph node metastases, whereas in the remaining case only two of four nodal metastases were positively stained. In no single case was a positive staining observed in a metastasis where the primary tumor gave a negative staining result. Five of the six cases with p53 mutations and fresh material available for cell culture were successfully grown in vitro and all had abnormal karyotypes.