We demonstrated previously a negative association of granulosa cell cocaine- and amphetamine-regulated transcript (CARTPT) expression with follicle health status and inhibitory effects of the mature CARTPT peptide (CART) on follicle-stimulating hormone (FSH) signal transduction in vitro, resulting in reduced bovine granulosa cell CYP19A1 mRNA and estradiol production. The objectives of this study were to investigate temporal regulation of granulosa cell CARTPT expression (granulosa cell mRNA and follicular fluid CART peptide concentrations) during follicular waves, CART regulation of androstenedione production (precursor for estradiol biosynthesis) by thecal tissue collected at specific stages of a follicular wave, FSH regulation of granulosa cell CARTPT mRNA expression, and the ability of CART to inhibit granulosa cell estradiol production and CYP19A1 mRNA expression when administered in vivo. CART concentrations in healthy, estrogen-active follicles (estradiol greater than progesterone in follicular fluid) decreased after dominant follicle selection, and CARTPT mRNA was lower in healthy, estrogen-active versus estrogen-inactive atretic follicles (progesterone greater than estradiol) collected at the predeviation and early dominance stages. CART treatment reduced luteinizing hormone-induced androstenedione production by thecal tissue collected at predeviation and early dominance stages but not at later stages of a follicular wave. The FSH or insulin-like growth factor 1 treatment in vitro reduced granulosa cell CARTPT mRNA in a dose-dependent fashion. Administration of CART in vivo into follicles at the early dominance stage reduced follicular fluid estradiol concentrations and granulosa cell CYP19A1 mRNA. Collectively, results support a potential stage-specific regulatory role for CART in negative regulation of estradiol production associated with selection of the dominant follicle.