The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) enhanced human (h) GH- and ovine PRL-stimulated mitogenesis of the Nb2-11C clone of rat lymphoma cells. Maximal enhancement of 25% in the proliferation rate was achieved with 20 nM TPA. The enhancing effect was found at all levels of hGH (0.031-2.0 ng/ml), but was more pronounced at lower hormone concentrations. TPA alone had no effect on cell proliferation, and its activity was absolutely dependent on the simultaneous presence of the lactogenic hormones. We have analyzed the changes that occurred in the distribution of cells in different phases of the cell cycle during the first 22 h after exposure to hGH and measured the proliferation rate through 3 days. We have found that the mitogenic effect of hGH resulted from 1) an increase in the rate of G0/G1----S transition, 2) a decrease in the lag period required for entry into the S phase, and 3) an increase in the number of cells entering this transition. TPA enhanced all three effects. Binding of [125I]hGH was not affected by prior exposure to TPA, suggesting that the effect of TPA is at a postreceptor level. Proliferation of an autonomous clone of Nb2 cells that does not require lactogenic hormones for growth was not stimulated by TPA, although these cells bound [3H]TPA to the same extent as the PRL-dependent Nb2-11C clone.