The correlation between gene expression and DNA methylation is well established. In the present study we examined DNA methylation as a potential regulatory mechanism of class I histocompatibility antigen expression. We selected for study two cell clones from the same mouse tumour (GR9). One of them is H-2 class I-positive (A7) and the other negative (B9). Using five enzymes and three different probes, no rearrangement or deletion was detected in the MHC class I genes of the negative clone compared with the positive one. These results do not provide an explanation for the differences observed in the expression of class I antigens in A7 (Kd, Dd positive) and B9 (Kd, Dd negative). We then studied the DNA methylation status in both clones by Southern blot analysis. Clear differences between the Kd, Dd positive and negative clones were detected. Furthermore, both Kd and Dd were clearly expressed after culturing the H-2 class I negative clone with the demethylating agent 5-azacytidine. These results suggest that the methylation of certain MHC cytosines is a regulatory mechanism of H-2 class I gene expression.