The present study was undertaken to fractionate and identify the stimulatory factors within the mixed leukocyte culture (MLC) supernatant which induce a 150- to 1000-fold increase in the in vitro plaque-forming cell (PFC) response of T cell-deficient normal mouse adherent spleen cells. G-75 Sephadex gel filtration of the allogeneic supernatant revealed three distinct peaks of helper activity which were designated helper peaks (HP) 1, 2, and 3. The m.w. estimates for the three components were HP-1, 10 to 15,000 daltons; HP-2, 40≥77,000 daltons; and HP-3, 20 to 25,000 daltons. HP-1 and HP-2 were further investigated and found to contain minimal helper activity at their respective optimal concentrations when compared to the activity of the unfractionated supernatant. The two components did, however, exhibit the remarkable capacity to effect synergistically an almost complete restoration of the PFC response. Expression of the helper factor synergism was dependent on the absolute concentration of HP-1 in the cultures but relatively independent of the amount of HP-2. Furthermore, the capacity of HP-1 to synergize with HP-2 was also dependent on the introduction of HP-1 to the cultures during the first 20 hr of the incubation period, suggesting that this component was crucial to the initiation of the PFC response. The results suggest that HP-1 and HP-2 have distinct sites of action in the B cell activation pathway and that full expression of the PFC response is dependent on the lymphostimulatory events mediated by these two moieties.