Anterior pituitary glands from male adult rats were incubated in vitro in various media for a comparative study on the mechanism of action of LH releasing factor (LRF) and prolactin release inhibiting factor (PIF). The released hormones were measured by radioimmunoassay.Hypothalamic extract (HE) prepared from rat stalk-median eminence increased LH release and decreased prolactin release. Theophylline (5 X 1 0 - 3M) had little effect on basal LH release, but markedly enhanced the LRF effect of HE. Basal prolactin release was significantly promoted, and PIF effect was partially reversed by theophylline. Dibutyryl cAMP (10-3M) did not affect basal LH release, while it enhanced LH release when added together with theophylline. On the other hand, dibutyryl cAMP significantly increased basal prolactin release, and the effect was strengthened by theophylline. Low calcium medium did not affect basal LH release, while it markedly inhibited basal prolactin release. In low calcium medium, the effect of theophylline, especially that on prolactin release, was abolished. Sodium fluoride, only at a high concentration ( 1 0 - 2M), significantly increased LH release, while it failed to affect prolactin release. Potassium ion enhanced LH and prolactin release at concentrations of 6-9 X 10-2M, but no further increase was observed at higher concentration. The effects of high potassium medium and HE were additive in the case of LH release, and competitive on prolactin release. Theophylline potentiated the effect of high potassium on the hormonal release, especially on LH release. These results appear to indicate an involvement of the adenyl cyclase-phosphodiesterase system in the mechanism of action of LRF and PIF. {Endocrinology 92: 698, 1973)