This study examined the use of PGF(2α) and estradiol benzoate (EB) either with or without GnRH to synchronize estrus in dairy cows for timed artificial insemination (TAI) under field conditions. First, Holstein dairy cows with a corpus luteum (CL) received 500 μg cloprostenol and were then randomly allocated to three groups: no further treatment (control, n=236); treatment with 1 mg EB 56 h after cloprostenol (EB group, n=339); or treatment with 1 mg EB 56 h after cloprostenol followed by treatment with 100 μg gonadorelin 24 h later (EB + GnRH group, n=216). All cows received TAI 80 h after the cloprostenol injection. In a second experiment, Holstein dairy cows with a CL received 500 μg cloprostenol and were then randomly allocated to two groups: treatment with 2 mg EB 36 h later (EB group, n=284) or treatment with 2 mg EB 36 h after cloprostenol followed by 100 μg gonadorelin 24 h later (EB + GnRH group, n=229). All cows received TAI 24 h after the EB injection. Logistic analyses revealed that the odds ratio for the probability of pregnancy when 1 mg EB was administered 56 h following cloprostenol was 1.9 and 2.0 times (P