We have utilized an in vitro clonogenic assay of mouse and human marrow granulocytic progenitor cells to determine the cytotoxic effects on granulopoiesis of the chemotherapeutic agents 1-beta-D-arabinofuranosylcytosine (ara-C) and 6-thioguanine. Concentration- and time-dependent decrements to plateau levels of granulocytic colony-forming capacity occurred. The sequence of drug administration was important and synergistic cytotoxicity was noted when certain schedules of ara-C and 6-thioguanine combinations were used. Endotoxin-stimulated colony-forming cells had increased sensitivity to the in vitro ara-C exposure. High or intermittent doses of ara-C demonstrated enhanced cytotoxicity when short exposure times (1 to 8 hr) were utilized, whereas low doses were markedly cytotoxic with prolonged exposure (10 days). Normal and leukemic human colony-forming cells had similar susceptibility to the cytotoxic effects of ara-C. Exposure of granulocytic precursors to these drugs in vitro produced effects similar to those previously reported with in vivo drug administration. These techniques appear applicable for providing improved screening models to evaluate chemotherapeutic regimens for clinical use.