The effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the growth and differentiation of cultured human acute promyelocytic leukemia (HL60) cells have been studied using cells growing in a fully defined medium consisting of RPMI 1640 supplemented with selenium dioxide, insulin, and either transferrin or ferric citrate. High concentrations of TPA (greater than 1 nM) cause the expected inhibition of proliferation and induction of macrophage-like differentiation. In contrast, in cells deprived of insulin, which continue to grow at a slow rate, lower concentrations of TPA stimulate proliferation without inducing differentiation. A TPA concentration between 0.03 and 0.3 nM will approximately double the long-term rate of thymidine incorporation into DNA and the rate of increase in cell density. Low-TPA becomes progressively less able to stimulate further proliferation as the insulin concentration is increased and is virtually without effect on cells stimulated by an optimal insulin concentration (5 micrograms ml-1). Insulin itself stimulates proliferation to a greater extent than low-TPA, increasing the long-term rate of thymidine incorporation and the rate of increase in cell density by three- to fourfold. The ability of higher concentrations of TPA to induce differentiation is independent of the presence of insulin. Low-TPA also stimulates the short-term incorporation of thymidine (during a 1-h pulse after 1 or 2 days incubation) by three- to fourfold, as compared to a sevenfold stimulation by insulin. The proliferation response to low TPA concentrations provides a useful model for dissecting the signalling pathways that control cell proliferation following stimulation by insulin and activators of protein kinase C.