Several alkylating carcinogens were tested for their ability to inhibit [rat] DNA methylation in an in vitro assay. N-Methyl-N''-nitro-N-nitrosoguanidine (MNNG) was the only carcinogen studied that altered the methylase activity. MNNG apparently reacts with the DNA methylase protein and produces a mixed type of inhibition. Prevention of the MNNG effect by dithiothreitol and inhibition of the DNA methylase with iodoacetamide suggest that DNA methylase is a sulfhydryl-containing enzyme and that MNNG inactivates the enzyme by reacting with sulfhydryl groups.