Specific binding sites for prolactin have been detected in membrane preparations from the testis, epididymis, seminal vesicles and prostate of male rats. The binding was time and temperature dependent. In prostatic tissue the binding of prolactin was a saturable process with an association constant (Ka) of 3 X 10(9) M-1 and a binding capacity of 125 femtomoles per mg of protein. The binding of prolactin to the prostate was inhibited only by lactogenic hormones. In the testis the low binding of prolactin increased slightly from 20 to 70 days of age. On the other hand, in the prostate the highest specific binding was found in membrane preparation from 20-day-old rats while 270-day-old rats had only 10% as much specific binding. The administration of estrogen also lowered prolactin binding to prostatic membranes. Castration caused an even greater decrease in the binding of [125I] iodo PRL in the prostate whereas in the liver this procedure resulted in a major increase in the binding of labeled prolactin.