In mares, circulating immunoreactive inhibin concentrations increase during the follicular phase and decrease at the start of the LH surge. Thereafter, sharp increases in circulating immunoreactive inhibin concentrations, the 'ovulatory increase', are observed during ovulation. In the present study, the cellular sources and molecular form of ovarian inhibin were investigated to determine the mechanism responsible for this unique ovulatory increase. Three sizes of ovarian follicles (small, < 15 mm; medium, 15-30 mm; large, > 30 mm in diameter) were selected. Inhibin alpha-subunit was localized by immunohistochemistry to the granulosa cells of follicles of all sizes and the theca cells of large follicles, whereas inhibin betaA- and betaB-subunits were detected in the granulosa and theca cells of large follicles only. High concentrations of immunoreactive inhibin, inhibin pro-alphaC and inhibin A were detected in the follicular fluid of large follicles compared with small and medium follicles, whereas there were no significant differences in the concentrations of inhibin B in the follicular fluid of medium and large follicles. These results indicate that mature large follicles secrete large amounts of inhibins pro-alphaC and A, whereas small or medium follicles secrete small amounts of inhibins A, B and pro-alphaC. These findings also indicate that the large amount of inhibin pro-alphaC produced by the ovulatory follicle is the source of the ovulatory increase in the concentrations of circulatory immunoreactive inhibin observed during ovulation in