In normal human tissues, pepsinogen A mRNA was expressed only in the fundic mucosa of the stomach, whereas pepsinogen C mRNA was expressed in all regions of the stomach mucosa and also in the proximal duodenal mucosa. The distributions of these mRNAs were consistent with those of pepsinogens A and C in the gastroduodenal mucosa. Methylation analysis of DNAs from normal tissues with methylation-sensitive restriction enzymes, HpaII and HhaI, revealed that pepsinogen A and C genes are hypomethylated in tissues producing pepsinogens A and C, suggesting a role of DNA methylation in the regulation of the differential expression of the genes for the two human pepsinogens during normal differentiation. In stomach cancer tissues and cancer cell lines, the expressions of the pepsinogen genes were decreased or lost, in good accordance with their pepsinogen productions. No gross structural changes of the pepsinogen genes were observed in these cancers, but the methylation patterns of the pepsinogen genes were found to be altered in different ways in different cancers. The functional significance of the altered methylation is unknown; however, these results suggest that considerable heterogeneity of the methylation patterns occurs in human stomach cancers.