Cell surface expression of HLA class I/peptide complexes on tumor cells is a key step in the generation of T‐cell‐based immune responses. Several genetic defects underlying the lack of HLA class I expression have been characterized. Here we describe another molecular mechanism that accounts for the complete absence of HLA class I molecule expression in a tumor line (MSR3‐mel) derived from a melanoma patient. Hypermethylation of the MSR3‐mel DNA, specifically of HLA‐A and ‐B genes, was identified, which resulted in loss of HLA class I heavy chain transcription. Treatment of MSR3‐mel cells with the demethylating agent 5′‐aza‐2′‐deoxycytidine (DAC) allowed HLA‐A and ‐B transcription, restoring cell surface expression of HLA class I antigens and tumor cell recognition by MAGE‐specific cytotoxic T lymphocytes. The MSR3‐mel line was obtained from a metastatic lesion of a nonresponding patient undergoing MAGE‐3.A1 T‐cell‐based peptide immunotherapy. It is tempting to speculate that the hypermethylation‐induced lack of HLA class I expression is the cause of the impaired response to vaccination. This study provides the first evidence that DNA hypermethylation is used by human neoplastic cells to switch off HLA class I genes, thus providing a new route of escape from immune recognition. © 2001 Wiley‐Liss, Inc.