Two experiments were performed to test the hypothesis that administering PGF_2α concurrent with timed artificial insemination (AI) in lactating dairy cows would enhance pregnancy per AI (P/AI). In experiment 1, lactating Holstein cows (n = 297) in 1 herd were enrolled at non-pregnancy diagnosis (30 to 36 d after AI) to synchronize subsequent ovulation before AI. Cows were assigned randomly to receive (i.m.) 10 mg PGF_2α concurrent with AI (d 0; treatment) or no injection (control). Blood samples were collected on d −3, 0 and 13 to determine serum concentrations of progesterone. Ovaries were scanned via transrectal ultrasonography to determine follicle diameters (d −3), subsequent ovulation risk (d 13), and total volume of luteal tissue (d 13). Diagnosis of pregnancy occurred 32 and 80 d after AI. In experiment 1, ovulation risk post-AI exceeded 91% and did not differ (P = 0.96) between treatments. Treatment with PGF_2α at AI decreased (P = 0.05) double ovulation risk. In contrast, PGF_2α treatment numerically increased progesterone (5.4 ± 0.4 vs. 5.8 ± 0.4 ng/mL) and luteal tissue volume (7.8 ± 0.8 vs. 8.6 ± 0.8 ng/mL) on d 13 by 7.4% (P = 0.27) or 10.3% (P = 0.16) in control and treated cows, respectively. Pregnancy per AI at d 32 and 80 was numerically increased by 16.7% (P = 0.47) and 22.3% (P = 0.30), respectively. Cows with progesterone >0.5 ng/mL at timed AI had reduced (P < 0.001) ovulation risk and tended (P < 0.10) to have poorer P/AI; both unaffected by treatment. In experiment 2, lactating dairy cows (n = 1,066) in 2 other herds were enrolled after unsuccessful first or later inseminations, and assigned randomly to treatment or control as described in experiment 1. Initial (d 35) and confirmed (d 65) pregnancy diagnosis revealed no differences in P/AI or embryonic survival. Pregnancy per AI was greater (P < 0.05) in primiparous cows (by 21 to 24%), first-service cows (by 22 to 26%), and between herds (by 28 to 36%) at both pregnancy diagnoses. Pregnancy loss was greater (P = 0.04) for cows inseminated at first (10%) vs. later services (5.2%). We conclude that treatment with PGF_2α concurrent with timed AI did not improve pregnancy per AI or embryo survival in lactating dairy cows.