We characterized the enzymes that catalyze the deiodination of T₄ to T₃ in the male reproductive tract. Testis (T), epididymis (EPI), seminal vesicles (SV), prostate (P), bulbourethral glands (BG), 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 P, the enzymatic activity was completely inhibited by 1 mM 6-n-propyl-2-thiouracil (PTU), whereas in the other tissues the inhibition was partial (50%). The high susceptibility to PTU inhibition, a ping-pong kinetic pattern, and low cofactor (Km for DTT = 0.7 mM) and high substrate (Km for rT₃ = 0.4 µM) 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 17--estradiol. Our data also showed a direct association between D1 and a functional epidydimys marker, the neutral -glucosidase enzyme, suggesting that local generation of T₃ 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.