The object of this study was to examine the effect of porcine follicular fluid (PFF) alone or in combination with estradiol (E2) on pulsatile LH release during the interval between the mornings of diestrus 2 (D2) and proestrus in the rat. Steroids were removed from PFF by charcoal extraction. Preliminary studies indicated that 1 ml PFF given intraperitoneally suppressed FSH secretion for up to 15 h, with an onset of action between 3 and 4h and maximal suppression between 6 and 9 h. In subsequent experiments, six groups of animals were bled continuously for 3 h between 07.30 and 10.30 h at a rate of 50 µl whole blood/5 min: group 1 was bled on D2; group 2 was sham ovariectomized on D2 (08.30–09.30 h), immediately implanted with an empty capsule, given saline at 12.00 and 24.00 h, and bled on proestrous AM; groups 3–6 were ovariectomized on D2, implanted with an empty or E2 capsule, given 1 ml saline or PFF at 12.00 and 24.00 h, and bled 24 h following ovariectomy (OVX). Between D2 and proestrus plasma E2 levels increased, and there was no change in any parameter of pulsatile LH release. However, OVX on D2 reduced plasma E2 levels and increased mean blood LH levels above proestrous values due to increases in LH pulse amplitude and frequency. Restoration of physiological proestrous levels of E2 reduced the increase in mean blood LH levels, by lowering pulse frequency to proestrous values and by greatly reducing pulse amplitude. However, LH pulse amplitude and mean blood LH levels were still higher than values on proestrus. PFF alone produced no alteration in any parameter of pulsatile LH release compared with saline-treated animals. However, administration of PFF to E2-implanted rats reduced LH pulse amplitude and mean blood LH levels to proestrous morning values. PFF also suppressed the OVX-induced increase in plasma FSH levels in empty- or E2-capsule implanted rats. Moreover, PFF given to E2-implanted rats significantly reduced the increase in mean blood LH levels seen in response to LHRH in E2, saline-treated animals. These experiments (1) confirm our previous report on the restraining action of E2 on LH pulse amplitude and frequency between the mornings of D2 and proestrus; (2) demonstrate that PFF contains a nonsteroidal substance with the ability to synergize with E2 in the regulation of LH pulse amplitude during this 24-hour interval, and (3) indicate that this restraining action of PFF is exerted, at least in part, at the pituitary l