Fertility to timed AI (TAI) is profoundly affected by progesterone (P4) levels during hormonal synchronization protocols. Holstein-Friesian dairy cows managed in a seasonal-calving, pasture-based production system were randomly assigned to 2 treatments to manipulate P4 before TAI during growth of the preovulatory follicle. Cows in the first treatment (High P4; n=30) were submitted to a Double-Ovsynch protocol {Pre-Ovsynch [GnRH; 7 d, PGF2α; 3 d, GnRH] followed 7 d later by Breeding-Ovsynch [GnRH (G1); 7 d PGF2α; 24 h, PGF2α; 32 h, GnRH (G2); 16 h, TAI]}. Cows in the second treatment (n=30; Low P4) received the same Double-Ovsynch protocol but with an additional PGF2α treatment 24 h after G1. Overall, synchronization rate did not differ between treatments and was 92% (55/60). Unexpectedly, 37% of Low P4 cows were detected in estrus ~24 h before scheduled TAI and were inseminated ~16 h before scheduled TAI. Overall, P4 did not differ between treatments at G1, whereas High P4 cows had greater P4 concentrations at PGF2α and G2 than Low P4 cows. High P4 cows had the smallest mean follicle diameter at G2, whereas Low P4 cows with no estrus before TAI had intermediate mean follicle diameter at G2, and Low P4 cows with estrus before TAI had the largest mean follicle diameter. Low P4 cows with estrus before TAI had larger corpora lutea 15 d after TAI than Low P4 cows without estrus before TAI or High P4 cows. In accordance with corpus luteum size on d 15, High P4 cows and Low P4 cows without estrus before TAI had lower P4 from 4 to 46 d after TAI than Low P4 cows with estrus before TAI. Relative mRNA levels of the interferon-stimulated genes ISG15, MX1, MX2, and OAS1 were greater for Low P4 than for High P4 cows, whereas relative mRNA levels of RTP4 were greater for High P4 than for Low P4 cows 18 d after TAI. Treatment did not affect plasma pregnancy-associated glycoprotein concentrations after TAI; however, pregnancy-associated glycoprotein concentrations were affected by pregnancy status and parity. Treatment did not affect pregnancy per artificial insemination at 29, 39, or 60 d after TAI, and no pregnancy losses were observed from 39 to 60 d after TAI. We concluded that (1) Low P4 cows were more likely to express estrus than High P4 cows; (2) the subpopulation of Low P4 cows that expressed estrus had larger preovulatory follicles and greater P4 concentrations after TAI; and (3) regardless of estrus before TAI, all Low P4 cows had greater mRNA expression for 5 of 6 interferon-stimulated genes than High P4 cows 18 d after TAI.