Hypophysectomy of mice during the last half of pregnancy results in an increase in the maternal serum mouse placental lactogen-II (mPL-II) concentration. The present study was undertaken to isolate and identify the pituitary factors that regulate mPL-II. Administration of pituitary extracts from virgin and day 12 pregnant mice (five pituitaries per mouse, every 8 h, sc) and bilateral pituitary implants under the kidney capsule significantly inhibited the increase in the mPL-II concentration that occurs within 24 h after hypophysectomy on day 12 of pregnancy, which indicated that the pituitary secretes factors that regulate mPL-II. To characterize these factors biochemically, pituitaries from virgin female mice were incubated for 14 days, and the culture medium was pooled, concentrated, and then fractionated by gel exclusion chromatography on Sephadex G-100, followed by ion exchange chromatography on DEAE. The fraction from DEAE chromatography that contained predominantly mouse (m) GH as well as a very small amount of a substance with an approximate mol wt of 23K suppressed the posthypophysectomy rise in serum mPL-II. To determine whether purified PRL, GH, and the unknown 23K substance present in the active fraction from the DEAE column could suppress the posthypophysectomy rise in serum mPL-II, repeated injections and constant infusion of these substances were carried out in hypophysectomized mice. Repeated injections of purified mPRL and bovine (b) PRL and constant infusion of mPRL were without effect. Repeated injection of mGH at doses of 1 and 10 micrograms/mouse or of bGH at doses of 50, 150, and 450 micrograms/mouse were also not effective, but bGH injections at a dose of 5 mg/mouse suppressed the posthypophysectomy rise in serum mPL-II. Subsequently, two pools of mGH were prepared by ion exchange HPLC; mGH-A contained predominantly mGH as well as a very small amount of an unidentified 23K mol wt substance, and mGH-B contained only mGH. When mGH-A and mGH-B were infused at a constant rate into hypophysectomized pregnant mice, both suppressed serum mPL-II concentrations; there was no difference in activity between mGH-A and mGH-B. These results indicate that mGH, but not mPRL, regulates the serum PL-II concentration in the mouse. mPL-II secretion is under the inhibitory control of mGH, the concentration of which increases rapidly at the beginning of the last half of pregnancy.