We have previously demonstrated that human rIL-12 alone can augment the development of cytotoxic activity in stimulated CD8+ T cells. The present study was undertaken to examine the interactions of rIL-7 and rIL-12 on human peripheral blood T cell activation and CTL differentiation. Purified T lymphocytes were pulsed overnight with immobilized alpha-CD3 and then cultured for 3 additional days with IL-7 and/or IL-12. The combination of IL-7 and IL-12 synergistically enhanced the proliferation of either fresh CD3+ T cells or an IL-2-dependent CD4+ T cell line, Kit-225-K6. This synergy was seen on both subsets of T cells; however, CD8+ T cells were usually more responsive to IL-7 and IL-12 at lower concentrations than were CD4+ T cells. Furthermore, these cytokines additively/synergistically augmented the cytotoxic activity of CD8+ T cells. Abs to IL-2 and IL-2R alpha blocked the synergistic effect on proliferation of CD4+ T cells, but had a minimal effect on the synergistic response of the proliferative and cytotoxic activity of CD8+ T cells. Examination of the effects of IL-7 and IL-12 on the expression of IL-12 receptor on T cells revealed an increase in the subunit of IL-12R by IL-7 as determined by flow cytometric analysis. We analyzed the effects on IFN-gamma production by CD8+ T cells and found that IL-7 alone did not induce detectable levels of IFN-gamma production but together with IL-12 it synergistically enhanced the production of IFN-gamma. We also found that IFN-gamma was probably not required for enhanced CTL activity of CD8+ T cells, because Ab to human IFN-gamma did not block additive/synergistic effects of either cytokine. The synergistic stimulatory activity of IL-7 and IL-12 may be of significance in vivo and may provide an alternative mechanism of stimulating T cells for use in immunotherapy.