The role of L3T4 antigens in the concanavalin A (Con A) response of regenerating spleen cells (Cy-SCs) after cyclophosphamide (Cy) treatment was studied. Anti-L3T4 monoclonal antibodies (Mabs) markedly inhibited the Con A response of the regenerating Cy-SCs, which do not require Ia molecules expressed on accessory cells (ACs) for Con A activation. However, the Con A response of normal spleen cells (N-SCs), which do require Ia molecules on ACs, was not inhibited by the same Mabs, although the Con A response of N-SCs, as well as that of Cy-SCs, was demonstrated to be mediated by L3T4+ T cells. The optimal times for the inhibitory effect of anti-L3T4 Mab was 7 days after Cy treatment, when the number of spleen cells increased to a maximum following a regenerative phase. Its inhibitory effect was reduced by high concentrations of Con A, and was restricted to the early phase of the Con A response. A short time exposure of the Cy-SCs to the anti-L3T4 Mabs was sufficient to decrease the response to Con A. Our results cannot explain the hypothesis that the L3T4 molecule functions solely by interacting with non-polymorphic parts of Ia molecules on ACs. Taken together, these results and those of other groups of investigators suggest that Con A-induced T-cell activation may be mediated by at least two or more interaction mechanisms involving either Ia or L3T4 molecules. Firstly, normal L3T4+ T cells may mainly interact with Con A involving self Ia molecules on the ACs. The extent of this interaction is sufficient to induce T-cell activation, and then does not need another L3T4 molecule. Secondly, the regenerating L3T4+ T cells may usually interact with the cell surface antigens of other T cells, including L3T4+, by the binding of both cell surface molecules to Con A in the absence of ACs, and then transmit a signal for T-cell activation. Anti-L3T4 Mabs may exert inhibitory effects somewhere in this process.