Immunohistochemistry using an antiserum raised against the synthetic follistatin peptide (residues 123-134) was used, in the present study, to detect the stage-specific appearance of immunoreactive follistatin in the rat testis. Follistatin immunoreactivity was not found in Sertoli and Leydig cells, while it was clearly detected in spermatogenic cells. Follistatin-like immunoreactivity was detected in the cytoplasm and nucleus of late pachytene spermatocytes. Although the reaction in the cytoplasm disappeared after meiosis, it continued to be intense in the nucleus from pachytene spermatocytes to round spermatids. This finding indicated that follistatin or its closely related peptide produced in late pachytene spermatocytes migrates from the cytoplasm to the nucleus. We subjected rat testis homogenate to affinity chromatography on a sulfate-cellulofine and anti-follistatin Cys (123-134)-Affi-Gel Hz column followed by reverse-phase HPLC and analyzed the resulting fractions by Western blotting using follistatin antiserum. Three major bands at 57, 45 and 39 kDa or four bands at 52, 44, 39 and 34 kDa were detected in crude preparations from rat testis homogenate, under reducing or non-reducing SDS-PAGE respectively. The protein from rat testis, which was recognized by anti-follistatin (123-134) antiserum, exhibited a characteristic pattern for follistatin on SDS-PAGE, i.e. slower migration under reducing conditions than under non-reducing conditions, suggesting that it was follistatin or its closely related protein. Follistatin or its closely related protein may be a stage-specific modulator of spermatogenesis. Since follistatin-like immunoreactivity was not found in oocytes in any stage of development from embryonic to adult rats, it may act in an event specific to spermatogenesis, such as nuclear condensation.