The role of inhibin, activin, and follistatin in the pathophysiology of polycystic ovary syndrome (PCOS) was investigated by examining the expression of human inhibin/activin subunit, follistatin, and type II activin receptor (ActRII and -IIB) messenger ribonucleic acid (mRNA) signals (via in situ hybridization) and encoded proteins (via immunocytochemistry) in ovarian follicles (n = 42) from 6 women diagnosed with PCOS. The localization patterns in cellular compartments were compared to those in small antral follicles of comparable size (3-7 mm; n = 40) from 17 normal human ovaries. In small antral follicles of both normal and PCOS ovaries, mRNA signals for all three subunits of inhibin and activin (alpha, beta a, and beta b) were expressed in granulosa cells, whereas in the thecal cell layer, only alpha-subunit mRNA was expressed. The relative intensity of the alpha-subunit mRNA signal was distinctly different in granulosa and thecal cells between PCOS and normal follicles; in small antral follicles of normal ovaries, the alpha-subunit mRNA signal was stronger in the granulosa cell layer than in the thecal cells, and the reverse was found in the polycystic follicles. A light follistatin mRNA signal was found in the granulosa cells of normal small antral follicles, but no follistatin mRNA was detected in any cell type of PCOS follicles. ActRII and -IIB mRNAs were not detected in any cell layer in either normal or PCOS follicles. There were no notable differences in the protein localization pattern of the inhibin/activin system between the PCOS and normal ovaries. In both types of follicles, follistatin and alpha-, beta a-, and beta b-subunit cytoplasmic staining were observed in granulosa cells, as were their corresponding messages, with the exception of the undetectable follistatin mRNA signal in the PCOS follicles. In both normal and PCOS follicles, follistatin and beta a-subunit cytoplasmic staining were occasionally found in thecal interna cells, with no corresponding localization of mRNA, and alpha-subunit protein was not detected in thecal cells despite the presence of the alpha-subunit mRNA. ActRII and -IIB protein localizations were not examined due to the lack of available antisera. These results suggest that granulosa cells of small antral follicles are less active in polycystic than in normal ovaries with respect to inhibin alpha-subunit and follistatin mRNA expression. A consequence of these differences could be an increase in the availability of activin, relative to inhibin, in the arrested follicles in PCOS.