The neuroactive steroid, 3α-hydroxy-4-pregnen-20-one (3αHP), is a metabolite of progesterone and a precursor of 3α-hydroxy-5α-pregnan-20-one (5αP3α; allopregnanolone). In addition to analgesic and anxiolytic effects by interaction with the GABA A receptor complex, 3αHP regulates pituitary FSH secretion by rapid non-genomic interaction with the Ca 2+ -driven cell signaling mechanisms. Since gonadectomy and adrenalectomy do not result in elimination of 3αHP, and since there is the possibility of paracrine and/or autocrine regulation of FSH release, the capacity of pituitary cells to regulate levels (by synthesis, metabolism, and storage) of 3αHP was examined. Anterior pituitaries from random cycling female rats were incubated, either as fragments or as cultured cells, for 1, 4 or 8 h with 3 H- or 14 C-labeled progesterone. The steroid metabolites were identified by thin-layer chromatography, autoradiography, high pressure liquid chromatography (HPLC), derivatization and GC/MS. Pituitary cells actively converted progesterone to 3αHP along with 5αP3α, 5α-pregnane-3,20-dione, 20α-hydroxy-5α-pregnan-3-one, 3β-hydroxy-5α-pregnan-20-one, 5α-pregnane-3α(β), 20α-diols, 20α-hydroxy-4-pregnen-3-one, and 4-pregnene-3α(β), 20α-diols. The results indicate the presence of the following steroidogenic enzymes in anterior pituitary cells: 3α-hydroxysteroid oxidoreductase (3α-HSO), 20α-HSO, 3β-HSO, and 5α-reductase. The activities of 5α-reductase and 3α-HSO were approximately equal and greatly exceeded those of the other enzymes. After 8 h of incubation with 100 ng progesterone per pituitary, about 20% of the progesterone was metabolized and 3.18 ng of 3αHP had been formed. The accumulation of 3αHP increased approximately linearly with the time of incubation. Metabolism studies using [1,2,6,7- 3 H]3αHP showed that pituitary cells convert about 29% and 8% of the 3αHP to progesterone and 5αP3α, respectively, in 2 h. Specific radioimmunoassays determined 11.6 and 7.5 ng of 3αHP per pituitary, respectively, in 25- and 40-day-old non-cycling female rats; these concentrations of 3αHP were about 2–3-fold greater than those of progesterone in the same pituitaries. In older (80–100 days old) cycling rats, the levels of 3αHP were about 9.4 and 18.6 ng/pituitary at 13.00 h and 22.00 h, respectively, on the day of proestrus, while the concomitant circulating levels were 13.7 and 5.4 ng/ml. The results indicate a marked capacity of rat pituitary cells to synthesize the neuroactive and FSH regulating steroid, 3αHP, from progesterone, and in turn to metabolize 3αHP to the neurosteroid, allopregnanolone, and to progesterone. The studies suggest cyclic biosynthetic and metabolic pathways for 3αHP and other steroids in the pituitary. They also indicate that the regulation of FSH secretion by 3αHP may be (in part, or in whole) via paracrine or autocrine mechanisms.