The present study examines the inhibitory action of progesterone (P) on prolactin (PRL) gene expression in the anterior pituitary of ovariectomized, estradiol (OVX+E) treated adult rats. A single injection of P (1 mg) was administered s.c. to OVX+E treated rats, and animals were killed at 3, 6, 9, and 36 h following P administration. Northern blot analysis showed that P suppressed the E-enhanced PRL mRNA level at 3, 6, and 9 h, but not at 36 h following P administration. When the second injection of P (1 mg) was given at 30 h after the first P, it again suppressed the E-induced PRL mRNA level, indicating that the inhibitory effect of P may not last until 36 h. The inhibitory action of P was dose-dependent, and the pretreatment of RU486 (100 micrograms/rat), a P receptor antagonist at 1 h before P injection partially restored PRL mRNA level which was inhibited by P. These data indicate that P plays a crucial role in the regulation of PRL gene expression in the rat pituitary.