Prothymosin alpha (ProT alpha), a 115-amino-acid thymic polypeptide, was tested for its effect on soluble antigen, allo- and auto-antigen-induced human T-cell proliferation. ProT alpha enhanced the secondary T-cell proliferative response to ovalbumin (OVA)- and keyhole limpet haemocyanin (KLH)-pulsed antigen-presenting cells (peripheral blood monocytes). Maximum enhancement (20-fold for OVA and 23-fold for KLH) occurred when suboptimal concentrations of either OVA or KLH were employed. Subset depletion experiments showed that the helper/inducer T-cell subpopulation was responsible for the observed enhancement. In the mixed lymphocyte reaction (MLR), ProT alpha enhanced autoantigen- (autoMLR; 9- to 14-fold) as well as the alloantigen- (alloMLR; 8- to 10-fold) induced T-cell proliferation when suboptimal ratios of the participating cells were used. Preincubation of the stimulating (autologous or allogeneic monocytes) with ProT alpha induced significantly higher T-cell proliferation in both primary and secondary MLR responses as compared to that induced by non-treated monocytes. In contrast, T lymphocytes pre-incubated with ProT alpha did not show enhanced proliferative activity when tested subsequently in the MLR. Suboptimal numbers of T cells exhibited high proliferative activity when pre-incubated with ProT alpha in the presence of autologous monocytes. These studies suggest that ProT alpha potentiates T-cell proliferative responses not directly, but via monocytes which are included in the cultures either as antigen-presenting cells or accessory and/or stimulator cells. The importance of ProT alpha in pathologically occurring defective cellular immune response systems discussed.