Levamisole has been reported to act as an immunological adjuvant. Experiments reported here on the effect of this agent on a variety of murine lymphoid culture systems were designed to gain an insight into its mechanism of action. We have found levamisole to be a weak mitogen for mouse spleen cells producing a dose related response which peaks at 48 hr in culture. The drug acted to augment the response of spleen cells to sub-optimal concentrations of concanavalin A, but had no unusual effect on the lipopolysaccharide stimulation of B-cell DNA synthesis in vitro. Levamisole was directly stimulatory on enriched T-cell populations and was found to have two actions: (1) to stimulate a subpopulation of T cells and (2) to augment the response of suboptimal mitogen concentrations of concanavalin A. In addition, we have found that murine thymocytes stimulated by concanavalin A were greatly potentiated in the presence of levamisole, but this population of cells could not be stimulated directly by the drug.