The effects of CGP 41251, a specific inhibitor of protein kinase C (PKC), its inactive derivative CGP 42700, and of staurosporine have been analyzed in vitro on T lymphocyte functions. The proliferation of fresh human peripheral blood lymphocytes stimulated with antigen (PPD) or anti-CD3 mAb was strongly inhibited by both staurosporine (IC50 < 0.01 microM) and CGP 41251 (IC50 = 0.092 microM) but not by the PKC inactive compound CGP 42700 (IC50 > 10 microM). Antigen-specific activation and proliferation of mouse lymph node T cells was inhibited by staurosporine and CGP 41251 with IC50 values of 0.008 and 0.05 microM, respectively. The inactive derivative caused 50% inhibition in this mouse T cell assay only at concentrations of 25 microM. In order to evaluate possible differential effects of PKC inhibitors on CD4+ T cell subsets, murine T helper cell type 1 (Th1) and type 2 (Th2) clones were used. The KLH-specific clone 9/6 secretes IL-2 and IFN-gamma (Th1), whereas clone 9A/B does not secrete these lymphokines but secretes IL-4 and IL-5 (Th2). It was found that CGP 41251 inhibited antigen-induced proliferation of both Th1 and Th2 equally well with an IC50 of 0.02 microM. Furthermore, CGP 41251 inhibited the IL-2 or IL-2 and IL-4-mediated growth of Th1 and Th2 cells (IC50, 0.08 and 0.02 microM, respectively). Moreover, CGP 41251, but not CGP 42700, inhibited antigen-specific killing of target cells by Th1 clones (IC50 = 0.2 microM), a phenomenon which does not require cell proliferation. When Th1 cells were preincubated with the compound, washed, and rested for 24 hr, they killed the target cells, whereas killing by similarly preincubated, washed, but not rested Th1 cells was inhibited. Thus, the inhibitory effect of CGP 41251 is reversible and excludes the possibility of inhibition due to toxicity at the IC50 dose given. The comparison of CGP 41251 and staurosporine showed that although CGP 41251 has lower activity, it is more specific and much less toxic than staurosporine. Thus, CGP 41251 is more suitable for PKC studies.