The ability of lymphokines to affect the development and differentiation of mouse thymocytes in vitro was evaluated in a carefully controlled 3-day organ culture system. Concanavalin A (Con A)-induced supernatant (SN) from the T-cell clone D10.G4, which contains high concentrations of interleukin-3 (IL-3), IL-4 and IL-5, but lacks IL-1, IL-2 and interferon (IFN), markedly increased the proportion of CD4+CD8- cells, and decreased the proportion of CD4+CD8+ cells. These effects were unaffected by dialysing the SN, showing them to be caused by macromolecular factors. Highly purified recombinant IL-3 and IL-4 could exert similar effects, rIL-3 and rIL-4 both increasing the proportion of CD4+CD8- cells, and rIL4 in addition reducing the proportion of CD4+CD8+ cells. In conjunction with the findings of other investigators, these results indicate that at least four lymphokines (IL-1, IL-2, IL-3 and IL-4) can control T-cell development in the thymus.