Treatment of ovine pituitary cell cultures with an acetone powder of porcine follicular fluid (APPFF; 50 micrograms/ml) decreased FSH secretion 60%, did not alter basal LH secretion, but increased by 2- to 3-fold the effectiveness of LHRH or D-Lys6-LHRH (10(-8) M) in releasing LH. Chromatography of APPFF on Matrex Gel Red A (MGRA) yielded a protein fraction (MGRA-IV) in which both FSH-inhibiting and LHRH-enhancing activities were enriched 8-fold. Both activities were destroyed by trypsin, but both were highly resistant to heat. The apparent mol wt of the active substance(s) was greater than 10,000. The LHRH-enhancing effect of MGRA-IV was reversible and declined, with an apparent half-life of 7 h, when MGRA-IV treatment was discontinued. There was too little estrogen in either APPFF or MGRA-IV to account for any of the activities. These results demonstrate that porcine follicular fluid contains LHRH-enhancing activity along with classical inhibin activity and that both activities may be linked in one molecule. These dual activities may be important in a number of species.