Maspin, a serine protease inhibitor, was originally reported as a tumor suppressor gene in breast and prostatic cancers. We examined maspin expression and/or the allele-specific methylation status in four gastric cancer cell lines, as well as normal, metaplastic, and cancerous epithelia obtained from 50 gastric cancer patients. Three gastric cancer cell lines exhibiting maspin overexpression showed hypomethylation at either both alleles or a haploid allele. Only one cell line (GCIY) was maspin-negative but maspin expression was reactivated after treatment with a demethylating agent, 5-aza-2'-deoxycytidine. Dense and diffuse immunoreactivity for maspin was observed in 40 (80%) of 50 gastric cancers and all gastric normal epithelia (GNE) with intestinal metaplasia (IM), but not in GNE without IM. We further analyzed the allele-specific methylation status in 10 of 50 cases subjected to immunohistochemistry by the crypt isolation technique followed by a bisulfite genome sequencing method. The maspin gene promoter region of all GNE without IM was hypermethylated on both alleles whereas those with IM frequently represented the haploid type of hypomethylation status. In six of seven gastric cancers in which crypt isolation was possible, demethylation frequently occurred and extended to both alleles. Maspin mRNA was amplified from GNE with IM and cancerous crypts but not from GNE without IM. These results suggest that demethylation at the maspin gene promoter disrupts the cell-type-specific gene repression in both GNE and gastric cancer.