Specific events in the ovary are dependent on gene expression in the tissue. By screening a rat ovarian granulosa cell cDNA library, a cDNA clone encoding a novel transcription factor-like protein containing a high-mobility group-box, referred to as granulosa cell high-mobility group-box protein-1 (GCX-1), was identified. The expression of GCX-1 is restricted to the hypothalamus, pituitary, testis, uterus, and ovary but was not detected in the adrenal gland. An in situ hybridization study revealed that the expression of GCX-1 was restricted to granulosa cell layers in early-stage follicles, and the expression was very low in large antral follicles and the corpus luteum, but localized expression in the testis or pituitary was not clear. Endogenous GCX-1 protein in the granulosa cells was identified by a Western blot analysis, and an analysis using the green fluorescence protein-GCX-1 fusion protein revealed that the GCX-1 protein was localized in the cell nucleus. GAL4 fusion protein-based assays demonstrated that GCX-1 is a potent transcriptional activator, and its putative transactivation domain was mapped to the region between amino acid residues 25 and 63 from the N terminus. These data strongly suggest that GCX-1 is likely a novel transcriptional activator that is exclusively expressed in reproductive tissues involving the hypothalamo-pituitary-gonadal axis, and functions as a specific regulator of follicular development, and may also participate in other specific events related to reproduction, particularly in the female.