Our objective was to evaluate the effect of delaying PRID removal during a 5-d PRID-synch protocol on expression of estrus before timed artificial insemination (TAI) and pregnancies per artificial insemination (P/AI) in Holstein heifers. Our hypothesis was that delaying PRID removal would decrease the percentage of heifers in estrus before scheduled TAI while not affecting P/AI thus reducing the need for estrus detection during the protocol. Nulliparous Holstein heifers at ~14 mo of age were randomly assigned to receive their first AI after synchronization with: 1) a 5-d PRID-synch protocol with PRID removal on Day 5 (PRID5; D0, GnRH + PRID; D5, PGF -PRID; D6, PGF; D8, GnRH + TAI; n = 230), or 2) a 5-d PRID-synch protocol with PRID removal on Day 6 (PRID6; D0, GnRH + PRID; D5, PGF; D6, PGF -PRID; D8, GnRH + TAI; n = 232). Heifers were inseminated if observed in estrus after PRID removal and before scheduled TAI, whereas heifers not observed in estrus received TAI as scheduled on d 8. Pregnancy diagnosis was performed using transrectal ultrasonography 32 and 67 d after AI. Data were analyzed by logistic regression using the GLIMMIX procedure of SAS. More (P < 0.01) PRID5 heifers received AI to estrus before scheduled TAI than PRID6 heifers (12% vs. 1%). Overall, P/AI did not differ between treatments 32 (P = 0.99, 55.7% vs 55.6% for PRID5 vs. PRID6 heifers, respectively) or 67 (P = 0.92, 54.8% vs. 54.3% for PRID5 vs. PRID6 heifers, respectively) d after AI. In addition, pregnancy loss between 32 and 67 d after AI did not differ (P = 0.65) between treatments (1.6% vs. 2.3% for PRID5 vs. PRID6 heifers, respectively). We conclude that delaying PRID removal by 24 h during a 5-d PRID-synch protocol decreased the incidence of estrus before scheduled TAI without affecting P/AI thereby decreasing the need for detection of estrus during the synchronization protocol. Supported by CEVA Santé Animale and USDA NIFA Hatch project 1006519