It is well known that deglycosylation of gonadotropins by enzymatic or chemical procedures or by deletion of sites for N-linked glycosylation produces antagonistic analogs which are able to interact strongly with the receptor and to inhibit binding of the wild-type hormone. In the present study, we analyzed the antagonistic properties of a naturally occurring basic follicle-stimulating hormone (FSH) charge isoform obtained after high-resolution chromatofocusing of human anterior pituitary glycoprotein extracts. Coincubation of increasing amounts of this isoform with a highly purified human pituitary FSH preparation or with recombinant human FSH at doses equivalent to their corresponding ED50 for estradiol and tissue-type plasminogen activator (tPA) production, inhibited FSH-induced estrogen production and tPA enzyme activity by cultured rat granulosa cells in a dose-dependent manner. These inhibitory effects were apparently exerted at steps following 3',5'-cyclic adenosine monophosphate (cAMP) formation and did not involve activation of the protein kinase C pathway since: (a) at low doses, this basic FSH isoform moderately increased FSH-induced cAMP production by cultured rat granulosa cells; (b) coincubation of the antagonist isoform with dibutyryl cAMP completely inhibited the effects of this cAMP analog on estrogen and tPA production; (c) the isoform was able to stimulate production of cAMP in a human fetal cell line expressing the recombinant human FSH receptor, and (d) the inhibitory effects of the isoform were not affected by staurosporine, a protein kinase C inhibitor. The effects of this isoform upon dibutyryl cAMP-induced estrogen and tPA production were blocked by the addition of a highly specific antibody directed against human FSH, further demonstrating that the antagonistic effects observed were due to FSH-like molecules. In contrast to the inhibitory effects exhibited by this basic FSH isoform, a more acidic FSH charge variant consistently acted as an agonist of pituitary and recombinant FSH on both estrogen production and induction of tPA enzyme activity. These results indicate that the anterior pituitary gland normally produces FSH isoforms which act as either agonists or antagonists of FSH at the target cell level.