Objectives were to evaluate the effects of inseminating cows observed in estrus following a PGF(2α)-based presynchronization protocol on reproductive and lactation performance. Weekly, Holstein cows (260 primiparous and 379 multiparous) were balanced by parity, body condition score at 3 d in milk (DIM), and previous lactation milk yield (multiparous cows) and assigned randomly to either of 2 reproductive programs. All cows received 2 injections of PGF(2α) at 35 and 49 DIM and a controlled internal drug release insert containing progesterone from 42 to 49 DIM. Cows assigned to the short voluntary waiting period (SVWP) treatment were inseminated if observed in estrus after the second injection of PGF(2α) of the presynchronization protocol, and those not inseminated were submitted to a timed artificial insemination (TAI) protocol (GnRH 62 DIM, PGF(2α) 69 DIM, GnRH 71 DIM, and TAI 72 DIM), whereas cows assigned to the long voluntary waiting period (LVWP) were all submitted to the TAI protocol and were TAI at 72 DIM. Plasma progesterone was determined at 35, 49, and 62 DIM for evaluation of interval from parturition to resumption of cyclicity. Pregnancy was diagnosed weekly at 32 and 60 d after first AI and at 42 d after subsequent inseminations. Percentage of SVWP cows inseminated in estrus was 58.9% and the interval from parturition to first AI was shorter for SVWP cows (64.7±0.4 vs. 74.2±0.5 DIM). Cows cyclic by 49 and 62 DIM were more likely to be inseminated in estrus than those anovular by 62 DIM (67.9, 61.0, and 32.8%, respectively) and cyclic cows by 49 and 62 DIM had shorter interval from parturition to first AI than anovular cows (62.6±0.7, 63.1±1.2, and 70.1±1.1 DIM). Treatment did not affect pregnancy per AI after first postpartum AI or the rate at which cows became pregnant. Cows that resumed cyclicity by 49 DIM had greater pregnancy per AI than cows still anovular by 62 DIM and became pregnant at a faster rate than cows that resumed cyclicity by 62 DIM and those still anovular by 62 DIM. Inseminating cows that displayed estrus after the presynchronization protocol did not affect reproductive performance compared with submission of 100% of cows to a TAI protocol.