This study was designed to evaluate whether decreasing circulating progesterone (P4) or increasing circulating estradiol-17beta (E2) near the time of artificial insemination (AI) in an Ovsynch protocol would increase pregnancies per AI (P/AI) in lactating dairy cows. Six hundred nineteen lactating Holstein cows (n = 772 inseminations) received Ovsynch (GnRH-7 d-PGF(2alpha)-56 h-GnRH-16 h-timed AI). Cows were randomized in a 2 x 2 factorial experiment of 4 treatments to receive or not receive 25 mg of PGF(2alpha) 24 h after the standard PGF(2alpha) of Ovsynch, or 0.5 mg of E2 at the time of the final GnRH of Ovsynch, or both. Blood samples were collected 24 h after normal PGF(2alpha) and at final GnRH to evaluate circulating P4. Ovarian ultrasound was done at final GnRH to determine preovulatory follicle size. Ovulation was confirmed by ultrasound 5 d after AI. Treatment with additional PGF(2alpha) increased the percentage of cows that had complete luteal regression (95.6%) compared with control cows (84.6%). In contrast, additional PGF(2alpha) had no detectable effect on P/AI (control = 41.5% vs. + PGF(2alpha) = 44.7%). Supplementation with E2 increased expression of estrus (84.4 vs. 37.2%), but had no effect on overall fertility and even tended to have a negative effect on fertility in cows that ovulated to the second GnRH (control = 51.5% vs. +E2 = 44.0%). Thus, additional treatments with PGF(2alpha) or E2 during Ovsynch can be used to increase synchronization and expression of estrus during Ovsynch, although the lack of improvement in fertility makes these treatments unwarranted.