To assess the effect of social isolation of growing rats on 24-h rhythmicity of circulating prolactin and growth hormone (GH) levels and submaxillary lymph node immune responses, male Wistar rats were either individually caged or kept in groups (4-5 animals per cage) for 30 d starting on d 35 of life. Plasma prolactin and GH levels, and submaxillary lymph node lymphocyte subset populations, interferon (IFN)-gamma release and mitogenic responses to concanavalin A (Con A) and lipopolysaccharide (LPS) were determined at six time intervals during the 24 h span. Social isolation brought about changes in mean values and 24-h pattern of plasma prolactin and GH levels and lymph node immune responses. After isolation, prolactin and GH mean values decreased, and lymph node T, B, non T-non B, CD8+, and CD4+-CD8+ cells augmented, whereas lymph node CD4+/CD8+ ratio, IFN-gamma release and mitogenic responses decreased. Social isolation resulted in disruption of 24 h rhythmicity of every immune parameter tested. CD4+/CD8+ ratio, IFN-gamma release and Concanavalin A (Con A) and lipopolysaccharide (LPS) responses correlated significantly with plasma prolactin or GH levels while T/B ratio correlated with plasma prolactin levels only. B, non T-non B, and CD4+-CD8+ cells correlated negatively with plasma prolactin. Modifications in mean value and 24-h rhythmicity of plasma prolactin and GH levels are presumably involved in the effect of social isolation on immune responsiveness.