Many studies have indicated a critical role for the oocyte growth factor, growth differentiation factor-9 (GDF9), in mammalian follicle development, but no information has been available concerning oviparous species. We cloned a cDNA for chicken GDF9 (162 base pairs) and used it in Northern blot analysis to identify a transcript at 1.7 kilobase in RNA isolated from the ovary of the hen. We also sequenced two full-length clones from a normalized chicken reproductive tract cDNA library. The cDNA clone for chicken GDF9 encodes a protein of approximately 449 amino acids and all six cysteine residues, and three of the four glycosylation sites are conserved with respect to mammalian GDF9. Chicken GDF9 is approximately 65% similar in the full-length cDNA sequence and 80% similar in amino acid sequence at the C-terminal region to GDF9 from several mammals. Quantitative polymerase chain reaction analysis (n = 5) indicated that GDF9 mRNA is greatest in follicles <1 mm in size compared with larger follicles or granulosa layers isolated from larger follicles. Immunocytochemical analysis showed strong expression of GDF9 in hen oocytes. In yolk-filled oocytes, the GDF9 was localized at the periphery of the oocyte. Finally, oocyte-conditioned medium (from <1-mm oocytes) resulted in a 2-fold increase in granulosa cell proliferation, which could be inhibited by preincubation of the conditioned medium with GDF9 antibody. These data suggest that GDF9 is present in the hen oocyte and that this factor is capable of enhancing granulosa cell proliferation, as has been demonstrated in mammals.