The objectives were the determine the effects of presynchronization with PGF2α 2 days before the 5-d timed artificial insemination (AI) protocol on ovarian responses and pregnancy per AI (P/AI) in dairy heifers. The hypothesis was that PGF2α would induce responsive heifers to be in proestrus at the initiation of the timed AI protocol, which was expected to improve ovulatory responses and P/AI. Weekly cohorts of Holstein heifers were blocked by age and, within block, randomly assigned to remain as control (CON; n = 255) or receive PGF2α on experiment Day -10 (PG; n = 255). All heifers were subjected to the 5-d timed AI protocol (Day -8, GnRH + intravaginal progesterone controlled internal drug release insert; Day -3, PGF2α and insert removal; Day -2, PGF2α; and Day 0, GnRH and AI). A subset of 22 blocks of heifers (n = 43) had their ovaries scanned by ultrasonography on experiment Days -8, -3, 0, and 2 and blood was sampled and analyzed for concentrations of progesterone on experiment Days -8, -7, -5, and -3. Pregnancy was diagnosed on experiment Days 32 and 60. On the day of the first GnRH of the timed AI protocol, PG heifers had smaller concentration of progesterone in plasma (CON = 4.5 ± 0.5 vs. PG = 0.5 ± 0.5 ng/mL), but larger follicular diameter (CON = 9.1 ± 0.5 vs. PG = 11.0 ± 0.5 mm), and a greater proportion of them had a follicle with at least 8.0 mm in diameter (CON = 61.9 vs. PG = 90.9%) than CON heifers, which resulted in increased ovulation to GnRH (CON = 19.0 vs. PG = 86.3%). Ovulation to the initial GnRH of the protocol increased as the concentration of progesterone in plasma decreased, from less than 20% when progesterone was greater than 5.0 ng/mL to more than 65% when progesterone was less than 1.0 ng/mL. More CON than PG heifers spontaenously ovulated before the day of timed AI. Detection of estrus on the day of timed AI did not differ between treatments (CON = 50.9 vs. PG = 46.6%), but P/AI on Days 32 (CON = 52.9 vs. PG = 61.1%) and 60 (CON = 49.0 vs. PG = 57.1%) after insemination tended to be greater for PG than CON; however, the benefit to presynchronization was observed in heifers inseminated with conventional (CON = 54.7 vs. PG = 67.4%), but not in heifers inseminated with sex-sorted semen (CON = 50.9 vs. PG = 52.8%). Administration of PGF2α 2 days before initiating the timed AI protocol induced heifers to be in proestrus, which enhanced ovulation to the initial GnRH and favored pregnancy per AI, particularly in heifers inseminated with conventional semen.