During pseudopregnancy in the rat, ovulation is suppressed and the corpora lutea are maintained, presumably under the influence of prolactin. Schwartz and Rothchild (1) observed a steady rise in the pituitary concentration of luteinizing hormone (LH) during pseudopregnancy, reflecting a reduction in LH release which prevented ovulation. The mammary gland, another target organ of prolactin, also grows during pseudopregnancy. The extent of growth is comparable to that during the first 11-1 2 days of pregnancy (2). Although a sharp decrease in pituitary prolactin concentration at the induction of pseudopregnancy has been reported (3), no data for an extended period of time are available. Thus, the present study was undertaken to determine the changes in pituitary prolactin content during the entire course of pseudopregnancy and its relationship to biochemical changes in the mammary gland.Materials and Methods. Adult rats of CFE strain obtained from Carworth Inc., New York City, N. Y. were used. Rats were maintained in a room lighted between 6 a.m. and 6 p.m. and received Agway rat and mouse pellets and water ad libitum. Stage of the estrous cycle was determined by daily examination of vaginal smears. Pseudopregnancy was induced by vibrating the cervix for 20-25 sec on the day of estrus with a glass rod attached to a dental drill (4). Rats that began cycling after stimulation were discarded.Rats in groups of eight were killed by decapitation immediately after stimulation, at 0.5, 2, 8, 16, and 24 hr, and at 2, 4, 6, 8, and 12 days after vibration of the cervix. One unstimulated group and one group showing proestrus smear after the termination of pseudopregnancy were also killed. The ante-* Supported by National Institutes of Health Grant HD 01940. rior pituitary glands were removed, weighed and stored at -20' for prolactin assay. The eight pituitaries from each group were pooled and prolactin was assayed by a modification of the method of Reece and Turner ( 5 ) using a four-point assay design. Low doses of pituitary (0.5 mg) and NIH-P-Ss ovine prolactinl (1.0 pg) were injected into right and left sides of the crop sacs, respectively, of eight adult pigeons (Homer breed), and high doses of pituitary (1.5 mg) and standard prolactin (3.0 pg) were injected similarly into eight additional birds. Potency and the several criteria of the validity of the assays were calculated by the method of Bliss (6).The six abdominoinguinal mammary glands were removed and analyzed for nucleic acids after separating the parenchyma from the surrounding connective tissue (7). The weights of the adrenal glands were recorded.Results. Anterior pituitary weights (Table I) increased slightly (not significant) 30 min after cervical vibration, remained high until 16 hr, and then gradually declined to a low value on day 10 of pseudopregnancy ( p < 0.01 ) . Adrenal weights declined 10% (p