The objective of this study was to determine if there were different in luteolysis and circulating estradiol (E2) concentrations in 7-d or 5-d Co-Synch with one or 2 doses of prostaglandin (PGF_2α). Dairy heifers (n = 60) were randomly assigned to receive 7-d or 5-d Co-Synch protocols. Animals received 100 µg GnRH and a CIDR insert on d 0 for 7 and 5 d, respectively. Heifers in 7-d Co-Synch received a single dose (25 mg) of PGF_2α at CIDR removal, 100 µg GnRH at 54 h after CIDR removal, and were inseminated concurrently, 54 h after CIDR removal. Heifers in 5-d Co-Synch received 25 mg PGF_2α at CIDR removal, the second dose 8 h later, 100 µg GnRH at 72 h after CIDR removal, and were inseminated concurrently, 72 h after CIDR removal. Blood collected on d 0, 7 or 5, and 9 or at the AI time was assayed for progesterone (P4) and E2. Defined luteolysis occurred when P4 was ≥1 ng/mL on d 7 or 5 and 72 or 54 h (d 9) was <1 ng/mL. Concentrations of P4 did not differ between groups. Luteolysis was not different between the groups [86.7% (25/30) vs. 90.0% (27/30)]. Diameter of follicle at the second GnRH did not differ (P > 0.05) between treatments (13.2 and 12.7 mm, respectively). Although treatments did not affect the conception rate (P > 0.05), concentrations of E2 were greater (P < 0.01) on d 9 or before fixed time AI in 5-d Co-Synch group than 7-d Co-Synch (6.5 vs. 4.7 pg/mL). These results provide evidence that 5-d Co-Synch with 2 doses of PGF_2α affects greater circulating E2 concentrations during preovulatory period and may increase conception success following a fixed time AI.