Objective: The effect of serotonin (5-hydroxytryptamine; 5HT) on the in vitro proliferation of mitogen-stimulated lymphocytes was studied in primary cultures of rat spleen cells. Methods: 5HT was added to the cultures 1 h prior to the mitogen, at final concentrations from 10^–13 up to 10^–2M. T and B cell mitogens (concanavalin A, pokeweed mitogen and lipopolysaccharide) were used at suboptimal and optimal concentrations. The cell proliferation was measured 24–72 h after the addition of mitogen. The effect of each 5HT concentration was studied on a group of 6–12 animals and was expressed as a percentage of the control values obtained with mitogen alone. Results: No significant effect of 5HT at concentrations from 10^–13 to 10^–5M was found. At concentrations of ≧10^–4M, a regular dose-dependent inhibition of the lymphocyte proliferation appeared, the concentration producing the half-maximal effect being 6 × 10^–4M. The observed suppression was not due to 5HT cytotoxicity toward spleen cells. Conclusion: With the experimental system used, we failed to confirm an immunostimulatory effect of 5HT in the range of concentrations of its receptor sensitivities or lower, but found a clear-cut immunoinhibitory effect at higher concentrations.