Thirteen lactating Holstein cows were assigned randomly to either a control or β-carotene (600 mg/d) treatment group to determine the effect of long-term β-carotene supplementation on releasable luteinizing hormone in response to gonadotropin-releasing hormone challenge. The experimental period was 28 wk. Milking was terminated at wk 16, ovariectomy performed at wk 20, and response to gonadotropin-releasing hormone determined at wk 27. Serum β-carotene concentrations reflected dietary intake and were higher in the cows fed β-carotene after 2 wk of supplementation and remained higher for the duration of the trial. Feeding supplemental β-carotene had no effect on circulating progesterone concentration, corpora lutea size or corpora lutea progesterone concentration, or basal concentrations of luteinizing hormone, frequency, and amplitude of luteinizing hormone pulses, or the release of luteinizing hormone in response to gonadotropin-releasing hormone. Thus, pituitary responsiveness to exogenous gonadotropin-releasing hormone was not affected by feeding supplemental β-carotene.