A Circadian rhythm in mammary gland response was observed in steroid primed Sprague‐Dawley rats which received daily injections of ovine prolactin for 5 days at either 0600, 1200, 1800, or 2400. Mammary gland weight (± SE) depended upon the time of day of prolactin injection; 5.97 ± 0.54 g (0600), 2.64 ± 0.46 g (1200), 2.75 ± 0.33 g (1800), 3.38 ± 0.36 g (2400). Consinor analysis of the response to prolactin data showed a highly significant 24‐h rhythm (P < 0.0001) with a mesor of 3.71 ± 0.24 g, an amplitude of 1.65 ± 0.66 g, and an acrophase of _77.95 ± 11.74 degrees (0512 ± 47 min.). The 105 female rats were ovariectomized at age 12 weeks and maintained in a room (22° C) on a 12‐h daily photoperiod (0600 to 1800). Steroid priming was produced by giving injections (i.p.) of2μg of 17 ß‐estradiol‐3‐benzoate and 6 mg of progesterone in 0.1 ml of corn oil for 20 days (0800 ‐ 0900), beginning 2 February). Prolactin (2 mg, 50 IU, in 0.25 ml of 0.9% saline) was injected (i.p.) at either 0600, 1200, 1800, or 2400 for 5 days (beginning 22 February) in the 4 groups of 12 rats. Saline injected and non‐injected groups were also included. The animals were killed 24 h after their last injection or at scheduled times for the non‐injected controls. The freshly dissected mammary glands were weighed.