Follistatin is a single-chain gonadal protein that specifically inhibits follicle-stimulating hormone release. By use of the recently characterized porcine follistatin cDNA as a probe to screen a human testis cDNA library and a genomic library, the structure of the complete human follistatin precursor as well as its genomic organization have been determined. Three of eight cDNA clones that were sequenced predicted a precursor with 344 amino acids, whereas the remaining five cDNA clones encoded a 317 amino acid precursor, resulting from alternative splicing ofthe precursor mRNA. Mature follistatins contain four contiguous domains that are encoded by precisely separated exons; three of the domains are highly similar to each other, as well as to human epidermal growth factor and human pancreatic secretory trypsin inhibitor. The genomic organization of the human follistatin is similar to that of the human epidermal growth factor gene and thus supports the notion of exon shuffling during evolution.The granulosa cells of the ovary secrete into the follicular fluid many proteins that can modify the secretion of pituitary follicle-stimulating hormone (FSH). Two such proteins, named inhibins A and B, which specifically inhibit the secretion of pituitary FSH, have been isolated and characterized from porcine (1-3) and bovine (4, 5) follicular fluid.The inhibins are heterodimeric proteins of M, 32,000, composed of a common glycosylated a subunit of Mr 18,000 and one of two similar , subunits of Mr 14,000 (1, 6). In addition, homo-and heterodimers of the /3 subunits of inhibins, named activins (7,8) or FRP (9), have also been identified in porcine follicular fluid, which can specifically stimulate the secretion of pituitary FSH. Throughout the purification of the inhibins and activins, we consistently observed one side fraction that also has FSH-release inhibitory activity and appeared unrelated to the inhibins. The fractions containing this activity have been purified to homogeneity to yield two glycosylated single-chain proteins of Mr 35,000 and 32,000, respectively (10). The two proteins have similar amino acid compositions and identical amino-terminal amino acid sequences and were named follistatins. Subsequently, three proteins with FSH release-inhibitory activity and having the same aminoterminal amino acid sequence as porcine follistatins were isolated from bovine follicular fluid by Robertson et al. (11). By use of amino acid sequence information derived from trypsin digestion of the Mr 35,000 protein, two populations of cDNA clones encoding, respectively, a follistatin precursor of 344 amino acids or its carboxyl-terminal truncated form with 317 residues were identified from a porcine ovarian cDNA library (12). The deduced precursor sequences bear no homology with the a, PA, and PB chains of the previously characterized porcine inhibins A and B. By use of the porcine cDNA as a probe, we report herein the cloning and sequencing of the human follistatin precursor as well as the determination of its genomic or...