The mitogenicity of 12-O-tetradecanoyl phorbol-13-acetate (TPA) for normal human peripheral blood mononuclear cells was investigated. TPA was a weak mitogen giving simulation indices in the range 2.5 to 10.5 at the optimum concentration (10 ng/ml) compared with 39 to 95 for phytohemagglutinin (PHA) at its optimum concentration (1 microgram/ml). No absolute requirement for a comitogen could be demonstrated, however TPA and PHA were synergistic in their action at low concentrations, and additive at optimum concentrations. Cell fractionation by rosetting with sheep erythrocytes showed that most of the proliferative response to TPA occurred in the T-cell fraction, however some proliferation of non-T cells was also observed. Surface marker studies showed that this could not have been due to residual T cells in the non-T fraction. A small number of monocytes was required for optimal proliferation of T cells in response to TPA. After a 3-day incubation with mitogen, the responding cell populations were tested for binding of a range of antibodies specific for T-cell (OKT3, OKT4, OKT8, and OKT11), "natural killer" (NK) cell (anti-Leu-7), monocyte (FMC17), and B-cell (anti-human immunoglobulin) surface markers. These experiments indicated that the responding cell types were T cells and B cells, but not NK cells or monocytes. Marked modulation of the antigen detected by OKT4, and to a lesser extent that detected by OKT3, in the presence of TPA precluded determination of which subpopulations of T cells proliferated in response to TPA. TPA was also tested for its ability to "maintain" activated T-cell blasts in a standard assay for interleukin 2 (IL-2). Mitogen-activated T cells were strongly responsive to TPA in this assay, but progressively lost responsiveness when maintained in crude IL-2 for about 2 weeks. Thus TPA does not have "maintenance" (i.e., IL-2-like) activity. However, small amounts of TPA acted synergistically with PHA in maintaining blast populations which were not responsive to TPA alone. This illustrates the importance of using long term IL-2-dependent cell lines for quantitation of IL-2 in supernatants prepared by stimulating T cells with these agents.