Murine lymphocytes have been shown previously to possess high-affinity specific receptors for the neuropeptide vasoactive intestinal peptide (VIP). This study examines the cellular basis for modulation of concanavalin A (Con A)-induced T-cell responses by this neuropeptide. VIP was most effective as an inhibitor when added at the initiation of the mitogen response. The loss of potency when VIP was added later in the response was accompanied by a decrease in the affinity of stimulated cells for the neuropeptide. The inhibitory influence of VIP was reversible if the neuropeptide was removed from stimulated cell cultures up to 6 hr after the initiation of stimulation. In contrast, VIP-mediated inhibition was fully developed once the stimulated cells had been exposed to the neuropeptide for 24 hr. The presence of VIP led to a decreased production of interleukin-2 (IL-2) by the stimulated lymphocytes, but did not affect the expression of Con A-induced suppressor cell activity by cultured lymphocytes. Studies of the effect of selective, complement-mediated killing of cells with Thy 1, L3T4 and Lyt 2 monoclonal antibodies showed that the majority of the VIP bound by the lymphocytes was accounted for by binding to L3T4+, Lyt 2- T cells. It was concluded that VIP exerts its influence over Con A-stimulated proliferation by selective regulation of T-cell subsets.