hCG has been reported to cause an inflammation-like effect in the testis, although the background and consequences of this phenomenon remain to be understood. This investigation reveals that a single injection of hCG (100 U) induces a transient surge in pro-inflammatory cytokine expression in the adult rat testis. Reverse transcriptase PCR analysis demonstrated onset of testicular expression of IL-1beta and IL-6 mRNA and increases in the levels of mRNA encoding the constitutively expressed cytokines IL-1alpha, IL-1 receptor antagonist, and tumor necrosis factor-alpha 4 h after hCG injection and a maximal response after 8-12 h. These increases were accompanied by a transient increase in testicular IL-1 bioactive protein. Twenty-four hours after administration of hCG, the levels of all cytokine mRNA had decreased, although most were still elevated above control. Immunohistochemical staining revealed that the IL-1beta protein was undetectable in normal testes but was seen to be localized to interstitial macrophages but not Leydig cells after hCG treatment. Testes devoid of Leydig cells after pretreatment with ethane dimethane sulphonate exhibited normal staining for interstitial macrophages but failed to respond to hCG with increases in IL-1beta mRNA and protein expression. We conclude that hCG induces testicular inflammation via local activation by Leydig cells of the production of pro-inflammatory cytokines by resident macrophages. It remains to be investigated whether the high-dose hCG regimens used for treatment of boys with cryptorchidism could induce similar increases of pro-inflammatory cytokines in the human testis and if such treatments could adversely affect future testicular function.