The objectives were to determine hypothalamic regulation of pulsatile luteinizing hormone (LH) secretion in female pigs and the biphasic feedback actions of estradiol-17β (E 2-17β). In the first study, the minimum effective dosage of E 2-17β that would induce estrus in ovariectomized gilts was determined to be 20 μg/kg body weight. In the second study, ovariectomized gilts were assigned randomly on day 0 to treatments: (a) hypophyseal stalk transection (HST), (b) cranial sham-operated control (SOC), and (c) unoperated control (UOC). On day 3, gilts from each group received a single i.m. injection of either E 2-17β (20 μg/kg body weight) or sesame oil. Blood was collected from an indwelling jugular cannula at 15 min intervals for 3 h before (day −2) and after treatment (day 2) from HST, SOC and UOC gilts. On day 3, blood was collected at 2 h intervals for 12 h after E 2-17β or sesame oil injection and at 4 h intervals thereafter for 108 h. Pulsatile LH secretion in all gilts 2 days after ovariectomy exhibited a frequency of 0.9±0.06 peaks/h, amplitude of 1.3±0.13 ng/ml, baseline of 0.8±0.07. Serum LH concentrations from SOC and UOC gilts were similar on day 2 and profiles did not differ from those on day −2. In HST gilts pulsatile LH release was abolished and mean LH concentration decreased compared with controls (0 versus 0.9±0.06 peaks/h and 0.77±0.03 versus 1.07±0.07 ng/ml, respectively; P<0.05). E 2-17β or sesame oil did not affect serum LH concentration in HST gilts, and LH remained constant throughout 120 h (0.7±0.07 ng/ml). In SOC and UOC control gilts, E 2-17β induced a 60% decrease ( P<0.05) in LH concentration within 12 h, and LH remained low until 48 h, then increased to peak values ( P<0.05) by 72 h, followed by a gradual decline to 120 h. Although pituitary weight decreased 31% in HST gilts compared with controls (228 versus 332 mg, P<0.05), an abundance of normal basophils was evident in coronal sections of the adenohypophysis of HST comparable to that seen in control gilts. The third and fourth studies determined that hourly i.v. infusions of LHRH (2 μg) and a second injection of E 2-17β 48 h after the first had no effect on the positive feedback action of estrogen in UOC. However, in HST gilts that received LHRH hourly, the first injection of E 2-17β decreased ( P<0.05) plasma LH concentrations while the second injection of E 2-17β failed to induce a positive response to estrogen. These results indicate that both pulsatile LH secretion and the biphasic feedback action of E 2-17β on LH secretion depend on hypothalamic regulatory mechanisms in the gilts. The isolated pituitary of HST gilts is capable of autonomous secretion of LH; E 2-17β will elicit direct negative feedback action on the isolated pituitary gland if the gonadotropes are supported by exogenous LHRH, but E 2-17β at high concentrations will not induce positive feedback in isolated pituitaries. Thus, the direct effect of E 2-17β on the pituitary of monkeys cannot be mimicked in pigs.