In previous studies, we demonstrated that the administration of a luteolytic dose of cloprostenol, followed by 750 IU hCG plus 3 mg estradiol benzoate (EB) 12 h later, synchronized estrus in cows in the luteal phase. Most cows were ready for service 48 h after the beginning of treatment. The objectives of this study were to evaluate the reproductive performance of lactating dairy cows treated with this method of estrus synchronization and to determine the effect of decreasing the hCG-EB dose on synchronization and pregnancy rates after timed AI. Data were obtained from cows first inseminated within an interval of 45 to 70 d postpartum. A total of 2,472 lactating dairy cows in their first to second lactation period were assigned to 4 groups. Cows estimated to be in the luteal phase by rectal palpation were treated with 500 mcg, im, of cloprostenol and assigned to 1 of 3 groups to be intramuscularly injected with hCG-EB 12 h later at the following doses: Group 1 (n=626), 250 IU of hCG and 1 mg of EB; Group 2 (n=592), 500 IU of hCG and 2 mg of EB; and Group 3 (n=664), 750 IU of hCG and 3 mg of EB. Cows displaying natural estrus were inseminated to serve as controls (n=590). The synchronized cows were inseminated 48 h after cloprostenol injection, and control animals visually determined to be in natural estrus during the morning or afternoon were inseminated the following morning. Pregnancy diagnosis was performed by rectal palpation at 34 to 40 d postinsemination. All synchronized cows showed estrous activity within 24 to 36 h after cloprostenol treatment and were considered to be ready for service 48 h after this treatment. There was a significant effect of treatment on the pregnancy rate, either to first AI or to 2 rounds of AI. The pregnancy rate in response to first or second rounds of AI was similar to control rates for cows in Groups 1 and 2, and lower than control rates in Group 3. Cows in Group 1 showed a higher pregnancy rate to first AI than those in Group 3 (P<0.0001), and a higher pregnancy rate to second AI rounds than cows in Groups 2 (P<0.02) and 3 (P<0.0001). The number of cows returning to estrus was unaffected by treatment. However, treatment significantly decreased (P<0.01) the time of return to estrus as the hCG-EB dose increased. These findings indicate that the lowest dose of hCG-EB treatment tested gave the overall best pregnancy results among the treated groups. Furthermore, the synchronization protocol used in this experiment allows effective AI management of lactating dairy cows without the need for estrus detection.