The objectives were to determine the effects of PGF(2alpha) treatment on the prevalence of subclinical endometritis (SCE) and fertility of dairy cows. A total of 406 Holstein cows (167 primiparous and 239 multiparous) from 5 herds were used. Uterine lavage for diagnosis of SCE, PGF(2alpha) treatment, evaluation of body condition scores (BCS), and collection of blood samples for estrous cyclicity determination were performed at 21, 35, and 49 d in milk (DIM). Polymorphonuclear cells (PMN) were quantified and thresholds for diagnosing SCE were selected by receiver operating characteristics analysis. Cows classified as having SCE at 35 DIM (>or=6.5% PMN) and 49 DIM (>or=4.0% PMN) had increased time to pregnancy; however, cows classified as having SCE at 21 DIM (>or=8.5% PMN) did not. Median days to pregnancy were delayed by 30 (151 vs. 121 d) and 40 (169 vs. 129) d for cows classified as having SCE at 35 and 49 DIM, respectively. Treatment with PGF(2alpha) did not affect the prevalence of SCE either at 35 (37.9 vs. 38.4%) or at 49 DIM (34.0 vs. 40.4%). Treatment with PGF(2alpha) did not affect time to first insemination (AI; median 76 DIM for cows treated with PGF(2alpha); 79 DIM for control. Nonetheless, PGF(2alpha) treatment increased pregnancy to first AI in all the cows (35.5 vs. 24.1%) and hazard ratio (HR) of pregnancy in cows with BCS <or=2.5 when all of the cows were evaluated (HR = 1.5; 95% confidence interval; CI = 1.1 to 2.0) and when only cows without SCE were evaluated (HR = 1.8; 95% CI = 1.2 to 2.7). Treatment with PGF(2alpha) did not affect the hazard of pregnancy in cows with SCE at 49 DIM (HR = 0.9; 95% CI = 0.6 to 1.3). In these farms, treatment with PGF(2alpha) did not affect SCE or time to first insemination, but did increase first-service pregnancy per AI and decreased time to pregnancy in cows with low BCS.