Spleen is a pivotal organ for regulating immune homeostasis. It has been shown that testosterone diminishes secretion of various inflammatory molecules under multiple conditions. However, the mechanisms of action of endogenous testosterone affecting immune responses in the spleen remain unknown. The aim of the present study was to evaluate the immune functions of the spleen in response to testosterone withdrawal after orchidectomy, and the impact of splenocytes on the bacterial endotoxin lipopolysaccharide (LPS)-induced secretion of inflammatory molecules. Male rats were divided into 3 groups, i.e. intact, orchidectomized (Orch) and orchidectomized plus replacement of testosterone propionate (TP) (Orch+TP). The Orch and Orch+TP rats underwent bilateral orchidectomy one week before TP replacement (2mg/kg body weight) or sesame oil in intact rats as controls for seven days. Orch resulted in a significant increase of spleen weight and basal secretion of nitric oxide (NO) from splenocytes. Additionally, LPS up-regulated cell proliferation and the secretion of tumor necrosis factor-alpha (TNF-α) in splenocytes of Orch rats. Orch further up-regulated phosphorylation of extracellular signal-regulated kinases. Interestingly, the plasma corticosterone concentration in the Orch group was higher than that in the intact and Orch+TP groups. Deficiency of testosterone-elevated TNF-α and NO secretion in response to LPS were confirmed in the rat splenocytes. Testosterone also significantly attenuated LPS-elicited release of TNF-α and NO in a dose-dependent manner. However, testosterone did not suppress splenic blastogenesis at doses in the 10(-10)-10(-7)M concentration range. In this context, testosterone might have a protective role against inflammatory responses in the spleen. The present study provides evidence to indicate that testosterone might modulate the immune system.