DNA in mammalian cells is enzymatically methylated at the 5-position of cytosine via S-adenosylmethionine and DNA methyltransferase. Several chemical carcinogens have been shown to inhibit this reaction, altering DNA methylation. We have been studying the mechanism by which carcinogens alter the methylation of DNA in order to better understand the cellular regulation of DNA methylase activity and to understand the role, if any, of DNA methylation in the carcinogenic process. We have utilized an in vitro assay for DNA methylase isolated from purified rat-liver nuclei. Ethionine, a liver carcinogen, given to rats 17 hr after partial hepatectomy inhibited the incorporation of [methyl-3H]-methionine into 5-methylcytosine residues of DNA. DNA isolated from these ethionine-treated rats was able to accept methyl groups from S-adenosylmethionine 8 times more than control DNA. It was further demonstrated that S-adenosylethionine competitively inhibited the DNA methylase resulting in hypomethylated DNA. N-Methyl-N-nitro-N-nitrosoguanidine reacted with the DNA methylase at the sulfhydryl sites inactivating the enzyme. Methylnitrosourea did not react directly with the methylase enzyme, but when reacted with DNA, the DNA methylase activity was inhibited by the carcinogen alkylated DNA. Sodium selenite also inhibited the enzyme non-competitively with a Ki of 6.7 microM. 5-Azacytidine prevented the 2 to 3 fold increase in DNA methylase seen 2 days following partial hepatectomy. All of these data with various carcinogens, altering DNA methylation by different mechanisms, support the hypothesis that DNA methylation plays a role in the initiation of carcinogenesis.