Lipopolysaccharide induces a synergistic uptake of tritiated thymidine in peripheral blood lymphocytes (PBL) when cultured with phytohemagglutinin or concanavalin A as compared to PBL incubated only with phytohemagglutinin or concanavalin A. In this study we investigated which subpopulations(s) of PBL is involved in this synergistic increase in deoxyribonucleic acid synthesis. Separation of PBL into sheep erythrocyte rosette-forming cells (T cells) and non-rosette-forming cells (B cells) showed that the T cells were responsible for the increased uptake of radiolabel. PBL and T cells had similar dose-response profiles and kinetic patterns. Paralleling this augmented tritiated thymidine uptake was an increase in the number of cells undergoing blast transformation. Delayed-addition experiments showed that the two mitogens must be added within 12 h of each other for maximal augmentation to occur. Finally, preincubation of T cells with lipopolysaccharide had no demonstrable effect on the amount of concanavalin A uptake by these cells. This model may provide unique information about the activation of human peripheral blood T cells compared to activation of these cells by one mitogen.