We characterized the enzymes that catalyze the deiodination of T(4) to T(3) in the male reproductive tract. Testis, epididymis (EPI), seminal vesicles, prostate, bulbourethral glands, spermatozoa, and semen were taken from sexually mature rats (300 g). Iodothyronine 5'-deiodinase (5'-D) activity was quantified by the radiolabeled-iodide-release method. 5'-D activity was 10-fold higher in EPI and semen than in the rest of the tissues. In EPI, semen, and prostate, the enzymatic activity was completely inhibited by 1 mm 6-n-propyl-2-thiouracil, whereas in the other tissues the inhibition was partial (50%). The high susceptibility to 6-n-propyl-2-thiouracil inhibition, a ping-pong kinetic pattern, and low cofactor (Michaelis Menten constant for dithiothreitol=0.7 mm) and high substrate (Michaelis Menten constant for reverse T(3)=0.4 microm) requirements indicate that EPI 5'-D corresponds to type 1 deiodinase (D1). Real-time RT-PCR amplification of D1 mRNA in this tissue confirms this conclusion. The highest EPI D1 expression occurred at the onset of puberty and sexual maturity, and in the adult, this activity was more abundant in corpus and caput than in the caudal region. EPI D1 expression was elevated under conditions of hyperthyroidism and with addition of 17beta-estradiol. Our data also showed a direct association between D1 and a functional epididymis marker, the neutral alpha-glucosidase enzyme, suggesting that local generation of T(3) could be associated with the development and function of EPI and/or spermatozoa maturation. Further studies are necessary to analyze the possible physiological relevance of 5'-D in the male reproductive system.