Xylazine is an adrenergic alpha(2) agonist, which is used in veterinary medicine as a sedative and anesthetic agent. In this work we found that xylazine administered in vivo at a dose of 2.5 mg/kg enhanced spleen cell proliferation and interleukin 2 (IL-2) production in cultures stimulated with concanavalin A (Con A), whereas doses of 10 and 25 mg/kg were inhibitory. A similar stimulatory (10 microM) and inhibitory (50-500 microM) effect on splenocyte proliferation and IL-2 production was observed in vitro. Clonidine, another alpha(2) adrenergic agonist, only had a stimulatory proliferative effect on splenocytes. Yohimbine, an alpha(2) adrenergic antagonist, abrogated the stimulatory action of both clonidine and xylazine, but not the suppressive proliferative activity of xylazine in vitro. The inhibited proliferation of splenocytes to Con A correlated with increased apoptosis of T cells. The apoptosis was not blocked by yohimbine or antibodies to Fas and Fas-L. N-Nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase, enhanced proliferation of splenocytes to Con A, partly abrogated the inhibitory effect of xylazine in the proliferation assay, and, only at high concentration (1000 microM), partly suppressed apoptosis of lymphocytes. The enhancing effect of L-NAME on the Con A-induced proliferation of splenocytes correlated with decreased NO production. However, decreased NO production observed in cultures with xylazine was followed by both decreased lymphocyte proliferation and apoptosis. Cumulatively, these results suggest that the immunosuppressive properties of xylazine on splenocytes in vitro are due to increased apoptosis of lymphocytes, predominantly involve NO-independent pathways, and are probably independent of its action through alpha(2) adrenoreceptors.