Traditionally, prostaglandin F2α (PGF_2α) has been included in short-term progesterone-based estrous synchronization (STPBES) protocols but its inclusion has been associated with a reduction in fertility at the synchronized estrus. To determine if there is an effect of timing of prostaglandin administration relative to the progesterone pre-treatment on fertility, ewes (n = 442) on 4 farms located in WV and PA were randomly assigned to receive controlled internal drug-releasing devices (CIDR-g, 0.3 g progesterone) for 5 d alone (n = 123; treatment 4), in combination with PGF_2α (5 mL Lutalyse; 25 mg Dinoprost) at CIDR insertion (n = 103; treatment 1) or removal (n = 100; treatment 2), or 25 mg PGF_2α alone (n = 116; treatment 3) before being joined with sexually mature rams. Data were analyzed using ANOVA with the model consisting of the main effects of treatments, farms and their interactions and additionally, least squares means for treatment effects were determined. Ewes receiving only PGF_2α had a lower estrous response than other treatments (58.7 ± 3.7% vs. 74.6 ± 37.6%; P < 0.0001) but tended to have a higher conception rate than ewes receiving combination treatments of PGF at insertion (78.8 ± 8.9% vs. 61.2 ± 4.9%; P = 0.08) or removal (78.8 ± 8.9% vs. 59.8 ± 5.0%; P = 0.06). PGF_2α ewes also had a significantly lower pregnancy rate to 1st service than ewes receiving the combination treatment with PGF_2α at insertion (41.6 ± 4.4% vs. 54.1 ± 4.5%; P < 0.05) and ewes receiving CIDR only (41.6 ± 4.4% vs. 58.5 ± 4.1%; P < 0.005). There were no effects of treatment on any other measures of reproductive performance. In conclusion, inclusion of PGF_2α at the beginning or end of progesterone pretreatment did not enhance synchrony of estrus or other reproductive outcomes. Furthermore, synchronization of estrus with a 5 d treatment of progesterone was sufficient to synchronize estrus with high fertility in ewes.