The objective was to evaluate effects of prostaglandin (PG) F2α administration at the beginning of a progesterone and estradiol-based treatment period on ovarian response and pregnancy rate (P/AI) in Bos taurus beef heifers. Heifers were treated with 500 μg of cloprostenol administered: a) in two half-doses (250 μg) at the time of progesterone device insertion and removal (two-PG), or b) in a single dose at the time of device removal (one-PG). In the two-PG group, administration of PG at device insertion resulted in lesser serum progesterone concentrations during the 7-d treatment period (P < 0.05). Additionally, diameter of the follicle from which ovulation occurred was greater, and ovulation occurred earlier in the two-PG compared with one-PG group (P < 0.05). Fixed-time artificial insemination (FTAI) was performed in 3479 heifers with two times for FTAI (48 compared with 54 h from device removal). There was no effect on P/AI percentage for the PG treatment or the time of FTAI. The FTAI at 54 h resulted in a greater P/AI percentage in the one-PG than two-PG group (70.5%, 253/359 and 63.5%, 254/400, respectively; P < 0.05). There were no differences between PG treatments when FTAI was performed at 48 h after device removal. In conclusion, the administration of PG at the time of intravaginal progesterone device insertion results in lesser progesterone concentrations, and an increased size of the follicle from which ovulation occurs, and the time of ovulation is earlier after device removal. The pregnancy rate was not affected by the PG administration at the time of device insertion. Copyright © 2019 Elsevier B.V. All rights reserved.