A plasmid containing the transforming Ha-ras gene and designated pT24-C3 was methylated in vitro using the sequence-specific bacterial methyltransferases HpaII and HhaI. Aliquots of the plasmid were methylated by the single enzymes or by the two enzymes simultaneously (double methylation). The transforming activity of the treated plasmids was assayed in the standard transfection assay on NIH-3T3 cells. Double methylation reduced the transforming activity of pT24-C3 about 80%, whereas treatment with the single methylating enzymes did not significantly affect the oncogene activity. Southern blot analysis of the transformants obtained with the methylated or mock-methylated pT24-C3 plasmids indicated in all the examined DNAs the presence of human Ha-ras sequences with methylation degrees consistent with the treatment of the plasmids. The Mr 21,000 oncogene protein p21 was also detected in several examined transformants. The DNA-demethylating agent 5-azacytidine restored the transforming activity of the double-methylated pT24-C3 upon 24 h incubation of transfected NIH-3T3 cells. Southern blot analysis showed integration of human Ha-ras with a methylation profile intermediate between the double-methylated and mock-methylated plasmids. It is suggested that DNA methylation of specific CG-containing target sites can affect the transforming activity of a human oncogene.