Groups of five Holstein heifers were hysterectomized on Day 10 of the estrous cycle, and beginning 21 days later were given daily injections of either oxytocin, estradiol, equine luteinizing hormone, adrenocorticotropin, or relaxin. Three additional groups were slaughtered at the end of six, 15, and 30-day control periods. A gradual decrease in luteal tissue weight, progesterone concentration, and 20-β-ol concentration occurred as the control period lengthened. Oxytocin injections (0.33 United States Pharmacopoeia (USP) units/kg body wt/day) for either 15 or 30 days had no luteolytic effect. Equine luteinizing hormone (20 mg/day for 15 days) significantly increased progesterone concentration, total progesterone, and total progestins. Estradiol (5 mg/day) administered to one group for six days and to another for 15 days significantly depressed 20-β-ol concentration, total progesterone, and total progestins. Estradiol injected into intact heifers on either Days 5 through 14, or 10 through 14 caused complete luteal regression. Relaxin injections into hysterectomized heifers for 15 days had no effect. Adrenocorticotropin (100 units/day) injected on Days 2 through 8 significantly decreased corpus luteum weight in intact Hereford heifers, but was ineffective when injected into hysterectomized heifers. These results indicate that a uterine luteolytic mechanism is necessary for complete luteal regression, or that the corpora lutea of hysterectomized cows are unresponsive to normal luteolytic mechanism (s). © 1969, American Dairy Science Association. All rights reserved.