Using an in vitro quantitative clonal culture technique of bone marrow granulocyte-macrophage progenitor cells (colony-forming units culture (CFU-c)), we studied the hematopoietic toxicity of azathioprine after unilateral and bilateral ureteral ligation, unilateral and bilateral nephrectomy, and splenectomy in C57BL/6 mice. Analysis of femoral bone marrow 18 hr after i.p. injection of azathioprine (300 mg/m2) revealed increased CFU-c toxicity in comparison to controls as follows: (1) bilateral ureteral ligation, P < 0.01; (2) bilateral nephrectomy, P < 0.01; (3) unilateral ureteral ligation, P > 0.05 < 0.1; (4) unilateral nephrectomy, P, not significant; and (5) splenectomy, P, not significant. Extrapolation from a dose-response curve for the toxicity of azathioprine on the bone marrow CFU-c indicated that bilateral ureteral ligation and bilateral nephrectomy had the effect of a 25 to 50% increase in the azathioprine dose. After bilateral ureteral ligation, serum granulocyte-macrophage colony-stimulating factor levels were increased and in vitro tritiated thymidine suicide studies showed an increased proliferative rate of the CFU-c. Since azathioprine is a predominantly cell cycle-specific agent, we suggest that increased sensitivity to azathioprine is related to the increased proliferative rate of the CFU-c. The findings provide a rationale for a clinical policy of azathioprine reduction when there is depressed renal function.