The objectives of this study were to evaluate replacing GnRH with hCG and the effects of 48-h calf removal (CR) on pregnancy rates of cows synchronized with the CO-Synch protocol. Suckled beef cows (n = 467) at two locations were assigned to treatment by breed, age, and calving date. Treatment included either GnRH with (n = 121) or without CR (n = 117) or hCG with (n = 115) or without CR (n = 114) using the CO-Synch protocol. On d 0 and 9, cows received either hCG (2,500 IU, i.m.) or GnRH (100 microg, i.m.), and on d 7 all cows received PGF2alpha (25 mg). At one location, blood samples were collected from all cows (n = 203) on d -14, -7, 0, 7, 9, and 16. Calves were removed on d 7 and returned on d 9 (48 h) from approximately half of the cows that received GnRH or hCG. Cows that were detected in estrus between d 6 and 9 were bred approximately 12 h later and received no further injections. Cows not observed in estrus by d 9 received a second injection of either GnRH or hCG and were timed-inseminated. The AI pregnancy rates for GnRH-treated cows with or without CR and hCG-treated cows with or without CR were 46, 49, 35, and 34%, respectively (P = 0.44). Pregnancy rates of cows differed by treatment x age interaction (P = 0.07), hormone (P = 0.09), and hormone x age (P = 0.01) but not by CR (P = 0.66) or CR x age (P = 0.33). Among 2-yr-olds, pregnancy rates were higher for cows treated with hCG without CR than for cows that received GnRH with calf removal, whereas cows treated with hCG with CR and GnRH without CR were intermediate. In addition hCG-treated 2-yr-olds had higher pregnancy rates than GnRH-treated 2-yr-olds regardless of calf presence, but the reverse was true for older cows. Overall, GnRH-treated cows (48%) had a higher (P = 0.09) pregnancy rate than hCG-treated cows (34%). Among anestrous cows, GnRH and hCG were similar (P = 0.40) in their ability to induce ovulation and corpus luteum formation after the first and second injections of GnRH (31 and 76%, respectively) or hCG (39 and 61%, respectively). More (P = 0.001) hCG-treated cows exhibited short estrous cycles following timed AI. We conclude that hCG is not a suitable replacement for GnRH to synchronize ovulation with the CO-Synch protocol in multiparous cows, although further evaluation among primiparous cows is warranted using hCG with the CO-Synch protocol.