The role of 17β-estradiol and progesterone in the in vivo synthesis of prostaglandins by the ovariectomized rat uterus was investigated. Seven days after ovariectomy, rats were treated with progesterone (2 mg x 2 days) then given a single injection of estradiol. Prostaglandin E (PGE) and F (PGF) were determined by RIA in uterine tissue and uterine vein plasma (UVP) at 0, 0.5, 1, 2, 4, 8, 12 and 24 h after the administration of 17β-estradiol (10 µg). PGF and PGE content (ng/uterus) remains constant from the time of estradiol administration to the latest time period studied. PGF and PGE concentrations (ng/100 mg uterine wt) show a gradual decline during the course of estrogen action which becomes significant only 12 and 24 h after estradiol administration and is correlated with the increase in uterine weight. Following progesterone administration, there is a significant increase in both uterine PGE content and concentration from ovariectomized levels. These results indicate that levels measured in UVP represent a true de novo synthesis of prostaglandins and could not be accounted for by the release of stored material into the uterine vein. PGF levels in UVP rose from control values of 2.42 ± 0.53 ng/ml to a maximum of 23.30 ± 4.96 ng/ml at 12 h and by 24 h had returned to basal levels. A comparison with animals not treated with progesterone revealed low levels of PGF in UVP at all time periods studied (0.5-12 h). PGE levels in UVP after estradiol administration to progesterone-treated and untreated rats were similar, although at 12 h, levels in progesterone-treated rats were significantly greater than the untreated group. Determinations of estradiol in peripheral plasma by RIA revealed a sharp peak 1 h after administration followed by a rapid decline to control values by 12 h.