We have developed a limiting dilution clonal assay for determining the frequency of 6-thioguanine-resistant (TGr) lymphocytes produced in rats by in vivo exposure to genotoxic agents. Spleen lymphocytes were isolated from female Fischer 344 rats and were cultured with 1 microgram/ml of phytohemagglutinin (PHA) for 40 hr. Northern blot analysis revealed that this procedure resulted in increased hprt and beta-actin mRNA synthesis. Conditions for optimum cloning were established by culturing four PHA-primed lymphocytes/well in 96-well round-bottom microtiter plates containing a medium supplemented with interleukin-2. These cultures also contained autologous and/or TK6 feeder cells inactivated with different doses of irradiation. Lymphocyte cloning efficiencies (CEs) were highest in plates containing both irradiated TK6 cells (5 x 10(3) cells/well; 90 Gy) and irradiated autologous feeder cells (5 x 10(4) cells/well; 50 Gy). CE did not depend on the number of primed lymphocytes/well when four or fewer target cells/well were cloned. To measure the effects of chemical mutagens on the frequency of TGr lymphocytes, rats were given a single i.p. injection of 0-150 mg/kg of N-ethyl-N-nitrosourea (ENU), a direct-acting alkylating agent, or 0-50 mg/kg of cyclophosphamide (CP), an indirect acting alkylating agent. Lymphocytes were isolated, primed, and cloned at 4 weeks after CP treatment and at 1, 2, 4 and 6 weeks after ENU treatment. CE in these cultures ranged from 12% to 27%. Cultures were also established for measuring CE in the presence of 6-thioguanine (TG) and these contained 5 x 10(3) irradiated TK6 cells and 5 x 10(4) primed rat lymphocytes/well. The frequency of TGr lymphocytes was calculated by correcting the CE in the presence of TG with the CE measured in its absence. ENU exposure produced a higher frequency of TGr lymphocytes than CP, but both chemicals produced a dose-dependent increase in TGr cells. In addition, the frequency of ENU-induced TGr lymphocytes increased with time after treatment. The TGr cells are presumed to be hprt mutants, but further analysis at the DNA level is required to establish this. The dose-dependent responses obtained with both ENU and CP treatments suggest that rat lymphocytes are sensitive to direct- and indirect-acting alkylating agents administered in vivo and that the rat lymphocyte assay is a useful complement to the in vivo/in vitro mouse assay for determining the mutagenicity of environmental toxicants.