The related members of the interleukin 6 (IL-6) family of cytokines, IL-6, leukemia inhibitory factor (LIF), and oncostatin M, act as major inflammatory mediators and induce the hepatic acute phase reaction. Normal parenchymal liver cells express the receptors for these cytokines, and these receptors activate, to a comparable level, the intracellular signaling through signal transducer and activator of transcription (STAT) proteins and extracellular-regulated kinase (ERK). In contrast, hepatoma cell lines show attenuated responsiveness to some of these cytokines that is correlated with lower expression of the corresponding ligand-binding receptor subunits. This study tests the hypothesis that the reduced expression of LIF receptor (LIFR) observed in hepatoma cells is mediated by altered DNA methylation. H-35 rat hepatoma cells that have a greatly reduced LIF responsiveness were treated with 5-aza-2′-deoxycytidine, an inhibitor of DNA methyltransferase. Surviving and proliferating cells showed reestablished expression of LIFR protein and function. Restriction landmark genomic scanning (RLGS) demonstrated genome-wide drug-induced alterations in DNA methylation status, with striking similarities in the demethylation pattern among independently derived clonal lines. Upon extended growth in the absence of 5-aza-2′-deoxycytidine, the cells exhibit partial reversion to pretreatment patterns. Demethylation and remethylation of the CpG island within the LIFR promoter that is active in normal liver cells correlate with increased and decreased usage of this promoter in H-35 cells. In conclusion, these results indicate that transformed liver cells frequently undergo epigenetic alterations that suppress LIFR gene expression and modify the responsiveness to this IL-6 type cytokine.