Mechanisms of antimicrobial protection in male reproductive organs are poorly understood. Defensins are antimicrobial peptides produced by many epithelial tissues. The goals of the present study were 1) to test the hypothesis that adult rat male reproductive organs express mRNA for rat β-defensin (RBD)-1 and RBD-2, 2) to examine if defensin mRNA expression in the testis and epididymis is induced by bacterial lipopolysaccharides (LPS), and 3) to investigate the effects of androgens on defensin mRNA expression in the epididymis. Total RNA from reproductive organs was analyzed by relative reverse transcription-polymerase chain reaction analysis. RBD-1 mRNA was detected in the testis. All segments of epididymis expressed equal levels of RBD-1 mRNA with higher expression than in the testis, whereas accessory sex glands showed expression equal to that in the testis. Expression of RBD-2 mRNA was primarily restricted to the penis. Effects of inflammation on defensin mRNA expression were examined in rats administered a unilateral injection of LPS from Pseudomonas aeruginosa or Escherichia coli. Expression of RBD-1 mRNA in the testis and epididymis was unaffected by LPS. To test the hypothesis that circulating androgens regulate RBD-1 mRNA expression in the epididymis, rats were subjected to bilateral orchiectomy (orch) or to orch plus a 3.5-cm implant containing testosterone. Expression of RBD-1 mRNA in the initial segment and caput was unchanged following 1-day orch but showed androgen-sensitive expression after 5 and 15 days. Expression of RBD-1 mRNA in corpus and cauda was not affected by orch. Results of this study suggest that RBD-1 may play an antimicrobial role in the testis and epididymis.