Gel electrophoresis was used in a comparative study of prolactin content in the hypophysis of rats of different age and sex, and at various stages of the estral cycle. The hormone level in the pubertal rats was twice or thrice greater than in the immature ones; it was by 16% less at the diestrus than at the estrus stage. There was no change in the hypophysis prolactin content in male rats at puberty. Ovariectomy was accompained by a sharp reduction of prolactin in the hypophysis. Replacing estradiol therapy increased the amount of prolactin in the hypophysis, without bringing it, however, to the level characteristic of intact rats. Estrogens are responsible for the maintenance of prolactin level, but apparently other factors influencing its content in the hypophysis also exist.