The genomic level of DNA cytosine methylation was significantly diminished in dividing BALB/3T3 A31 CL1-13 cells treated with several aromatic hydrocarbon carcinogens. Benzo[a]pyrene-induced decreases in DNA 5-methylcytosine levels were concentration dependent over the range of 0.1 to 1.0 microgram/ml when determined at the end of the 16 h treatment period. The enzymatic methylation of DNA cytosine residues was the most sensitive to inhibition 48 h after treatment as concentrations of benzo[a]pyrene as low as 0.033 micrograms/ml initiated significant reductions in 5-methylcytosine levels. This inhibition of DNA cytosine methylation may be mediated by alkylation of the DNA. Treatment of hemimethylated DNAs with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro benzo[a]pyrene (anti-BPDE), (+/-)-r,7-t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (syn-BPDE), and (+/-)-benzo[a]pyrene-4,5-epoxide inhibited the transfer of methyl moieties from [3H]S-adenosylmethionine to cytosine residues in the undermethylated strand in the presence of mouse spleen methyltransferase activity. The syn isomer of BPDE was the most potent in this action while the parent compound, benzo[a]pyrene, did not significantly decrease the methyl accepting abilities of treated DNAs. All chemical carcinogens that were tested and are known to transform BALB/3T3 cells initiated significant reductions in 5-methylcytosine formation by 48 h post-treatment. However, concentrations of some hydrocarbons which do not transform these cells 5- to 50-fold above effective concentrations of the transforming carcinogens also provided significant reductions in DNA cytosine methylation. Thus the inhibition of DNA methylation may be an important step in the initiation of oncogenic transformation of BALB/3T3 cells, but decreases in DNA 5-methylcytosine levels alone cannot account for the onset of this multi-step process.