The effects of vasoactive intestinal peptide (VIP) have been investigated on the mitogenic response of rabbit spleen cells. Specific binding of 125I-VIP to these mononuclear cells is rapid and saturable. Analysis of binding reveals two classes of binding sites, a class of high-affinity binding sites with KD = 0.93 +/- 0.11 nM and maximal binding capacity of 2000 +/- 560 sites/cell, and a class of low-affinity binding sites with KD = 225 +/- 58 nM and maximal binding capacity of 280,000 +/- 60,000 sites/cell. The VIP regulatory effect on mitogen-stimulated rabbit spleen cell proliferation appears to be time dependent and bimodal. When VIP was added simultaneously with mitogens, it induced an inhibition of the proliferative response. With concanavalin A (Con A) or pokeweed mitogen (PWM), addition of 10(-8) M VIP resulted in a maximal 30% inhibition of [3H]thymidine incorporation after 96 h of culture. This inhibitory effect was significant at concentrations from 10(-8)-10(-6) M and half-maximal inhibition was obtained with 1.2 x 10(-9) M VIP. By contrast, when rabbit spleen cells were preincubated for 18 h with VIP alone, the lymphocyte proliferative response to Con A was increased. However, this increase was mitogen-selective, since it was only observed when the T-cell mitogen Con A was used. The maximal response was obtained after 96 h of culture in the presence of Con A. The VIP stimulatory effect was dose-dependent with a maximal effect at 10(-7) M and a half-maximal effect at 1.7 x 10(-9) M VIP. The effect of VIP was also time-dependent, since a 6 h preincubation was sufficient to induce a significant increase in the proliferative response which was maximal after an 18 h preincubation.