The transforming growth factor-beta (TGF-beta) signaling pathway subserves an essential tumor suppressor function in various cell types. A heteromeric complex composed of TGF-beta type I (RI) and type II (RII) receptors is required for TGF-beta signaling. We have identified a subset of human gastric cancer cell lines which are insensitive to TGF-beta and which express a low level of TGF-beta type I receptor mRNA relative to a gastric cancer cell line which is highly responsive to TGF-beta. Using these cells, we show that hypermethylation of a CpG island in the 5' region of the TGF-beta RI gene provides another potentially important mechanism of escape from negative growth control by TGF-beta. This hypermethylation was found in four of five human gastric cancer cell lines and five out of 40 (12.5%) primary tumors examined. In human gastric cancer cell lines, treatment with the demethylating agent, 5-aza-2'-deoxycytidine, resulted in increased expression of the TGF-beta RI gene, but not the RII gene. Transient transfection of an RI expression vector into the TGF-beta resistant SNU-601 cell line restores TGF-beta responsiveness. These findings suggest that one of the mechanisms of escape from autocrine or paracrine growth control by TGF-beta during carcinogenesis could involve aberrant methylation of CpG islands in the 5' region of the TGF-beta RI gene.