The HPP1 gene was initially discovered because of its frequent hypermethylation in hyperplastic colon polyps, but it is also hypermethylated in colorectal adenomas and carcinomas. Expression of the DNA mismatch repair gene hMLH1 is diminished or absent in some hyperplastic polyps, and it has been suggested that HPP1 inactivation is associated with the progression of microsatellite-unstable colorectal tumors. We sought then to determine the prevalence of HPP1 silencing by DNA methylation in gastric adenocarcinomas and to define any association of this event with microsatellite instability (MSI) or hMLH1 hypermethylation. Thirty-two matched normal-gastric adenocarcinoma DNA pairs were studied for MSI status and hypermethylation of HPP1 and hMLH1. Five (100%) of 5 MSI-H tumors, 2 (50%) of 4 MSI-L tumors, and 8 (35%) of 23 MSS tumors demonstrated HPP1 hypermethylation. Eight (25%) of 32 tumors (5 of 5 MSI-H, 2 of 4 MSI-L, and 1 of 23 MSS) showed evidence of hMLH1 hypermethylation. All (8 of 8) of these hMLHI-methylated tumors demonstrated concomitant methylation at the HPP1 locus: there were no cases of hMLH1 methylation occurring in the absence of HPP1 methylation. In gastric adenocarcinoma, hypermethylation frequently targets HPP1. Moreover, hMLH1 hypermethylation occurs predominantly in the setting of HPP1 hypermethylation. HPP1 hypermethylation may represent an early event in mismatch repair-deficient gastric tumorigenesis.