In an attempt to define the antigonadotropic activity associated with the protein/peptide extracts of ovine pineals, melatonin- and steroid-free fractions obtained after subjecting crude ovine pineal acetone powder to soft gel chromatography were tested in three bioassay systems: (1) the conventional compensatory ovarian hypertrophy inhibition assay in rats, (2) the coupled bioassay in immature mice that enables one to distinguish factors acting at the level of the pituitary from those acting at the gonadal level, and (3) the classical in vitro pituitary cell culture assay for inhibin. The large molecular weight fraction, referred to as PI, behaved like a classical antigonadotropin as it suppressed compensatory ovarian hypertrophy following unilateral ovariectomy. Further, it not only lowered the basal and gonadotropin releasing hormone (GnRH)-stimulated release of follicle stimulating hormone (FSH) by the cultured pituitary cells, but also inhibited the human chorionic gonadotropin (hCG)-induced uterine weight gain in the coupled assay, thus acting like inhibin in these two assay systems. In addition, it showed immunological cross-reactivity with ovine testicular inhibin. The present results strongly support the view that inhibin-like activity may be the major player in what has so far been referred to as antigonadotropic activity.