In previous investigations into the mechanisms responsible for cell specificity in hepatocarcinogenesis, we have demonstrated that O6-methylguanine accumulates in the DNA of nonparenchymal cells (NPC) but is efficiently removed from hepatocellular DNA. O6-Alkylguanine may, therefore, be an important promutagenic lesion responsible for the induction of hepatic angiosarcomas after exposure to methylating agents, but other promutagenic DNA alkylation products--i.e., O4-alkylthymine--may be responsible for the initiation of hepatocellular carcinomas. F-344 male rats were provided drinking water containing diethylnitrosamine (DEN) at 40 ppm for 0, 2, 4, 8, 16, 28, 49, or 77 days, a regimen that selectively causes hepatocellular carcinomas. Hepatocytes and NPC were isolated by using low-speed differential centrifugation. DNA was purified by hydroxyapatite chromatography and hydrolyzed enzymatically, and O4-ethyldeoxythymidine (O4-EtdThd) and O6-ethyldeoxyguanosine (O6-EtdGuo) of hepatocyte and NPC DNA were quantitated by competitive radioimmunoassay using high-affinity monoclonal antibodies. O4-EtdThd accumulated in hepatocyte DNA during the first 28 days of DEN exposure, approximating a steady state at an O4-EtdThd-to-deoxythymidine molar ratio of approximately equal to 1 X 10(-5). This O4-EtdThd concentration was maintained from 28 to 77 days of DEN exposure. In contrast, O6-EtdGuo did not accumulate in hepatocyte DNA, its greatest concentration O6-EtdGuo-to-deoxyguanosine ratio (approximately equal to 3.7 X 10(-7) ) being detected after 2 days of exposure to DEN. O6-EtdGuo concentrations in hepatocyte DNA decreased with duration of exposure to DEN to an O6-EtdGuo-to-deoxyguanosine ratio of less than 2 X 10(-7) from 28 to 77 days. The data indicate that O4-EtdThd disappears from the DNA of hepatocytes less than 1/200th as fast as O6-EtdGuo. DNA from NPC contained approximately half as much O4-EtdThd as hepatocytes did, but greater than or equal to 2.5 times more O6-EtdGuo.