Retinoic acid (RA) has been shown to be an inducer of the terminal differentiation of several leukemia cell lines in vitro and in clinical trials to produce a high percentage of remissions in patients with acute promyelocytic leukemia. In an effort to increase the therapeutic efficacy of RA, we have measured the capacity of granulocyte colony-stimulating factor (G-CSF) to enhance the differentiation inducing activity of RA in WEHI-3B D+ monomyelocytic leukemia cells. Combinations of G-CSF and RA produced a supra-additive increase in the percentage of WEHI-3B D+ cells reducing nitro blue tetrazolium and expressing Mac-1 (CD11b) antigen on the cell surface, two markers of the mature state. In the presence of 50 ng/ml of G-CSF, which produced only 12% differentiation when used alone, 0.5 microM RA induced the same degree of cellular differentiation as the optimum concentration of RA (i.e. 7 microM) employed alone. The supra-additive differentiation produced by this combination was prevented by the presence of G-CSF monoclonal antibody in the culture medium, resulting in a degree of maturation comparable to that produced by the retinoid alone. When cells were sequentially exposed to G-CSF followed by RA, a much higher level of differentiation was obtained than when the order was reversed, suggesting that WEHI-3B D+ cells were primed to enter a differentiation pathway by G-CSF. The supra-additive terminal differentiation exhibited by the mixture of G-CSF and RA suggests that these agents should be evaluated for the therapeutic efficacy of the combination in patients with acute non-lymphocytic leukemia.