Ten closely related tumor-promoting and nonpromoting, proinflammatory phorbol derivatives were tested for stimulation of [3H]thymidine ( [3H]TdR) incorporation into human mononuclear cells. Co-mitogenic activity was assessed with maximally effective concentrations of phytohemagglutinin (PHA) or mixed-lymphocyte reactions (MLR) in the presence of phorbol esters. Tetradecanoyl phorbol acetate (TPA), two 4-deoxyphorbol esters, and two 12-deoxyphorbol monoesters stimulated [3H]TdR incorporation in a dose-related manner. Two established nonpromoting 12-deoxyphorbol diesters were also mitogenic, although less effective than TPA, and produced lower maximal responses. TPA, the 4-deoxyphorbol esters, the 12-deoxyphorbol monoesters, and the two nonpromoting diesters were able to increase MLR-induced incorporation to the same level. When conditions were used where PHA and phorbol esters were optimally mitogenic, inhibition resulted. With the exception of co-mitogenic activity of the nonpromoting diesters, the mitogenic, co-mitogenic, and PHA-inhibiting activities were correlatable. Phorbol, a 4 alpha-deoxyphorbol ester, and resiniferatoxin had no effects. Mitogenic activity of the phorbol esters was inhibited by dexamethasone, chloroquine, and p-bromophenacyl bromide. TPA, the 4-deoxyphorbol esters, and the 12-deoxyphorbol monoesters were resistant to inhibition of activity by cyclosporin A, whereas the noncorrelating diesters exhibited sensitivity to cyclosporin A inhibition comparable to that of PHA. MLR-induced [3H] TdR incorporation was susceptible to cyclosporin A, but the phorbol ester-enhanced responses were cyclosporin A-resistant.