GDF-8 is a new member of the TGF-beta superfamily which appears to be a negative regulator of skeletal muscle mass. Factors which regulate the biological activity of GDF-8 have not yet been identified. However, the biological activities of TGF-beta superfamily members, TGF-beta1, -beta2 and -beta3, can be inhibited by noncovalent association with TGF-beta1, -beta2 and beta3 propeptides cleaved from the amino-termini of the TGF-beta precursor proteins. In contrast, the propeptides of other TGF-beta superfamily members do not appear to be inhibitory. In this investigation, we demonstrate that purified recombinant GDF-8 propeptide associates with purified recombinant GDF-8 to form a noncovalent complex, as evidenced by size exclusion chromatography and chemical crosslinking analysis. Furthermore, we show that GDF-8 propeptide inhibits the biological activity of GDF-8 assayed on A204 rhabdomyosarcoma cells transfected with a (CAGA)12 reporter construct. Finally, we demonstrate that GDF-8 propeptide inhibits specific GDF-8 binding to L6 myoblast cells. Collectively, these data identify the GDF-8 propeptide as an inhibitor of GDF-8 biological activity.