The aim of this study was to evaluate luteolysis using three doses of PGF2α on Day 5 or Day 7 of the estrous cycle in nonlactating Nellore (Bos indicus) cows. Cows (n = 323) were assigned within date of estrus (Day 0 of estrous cycle) to receive 12.5, 25.0, or 50.0 mg of PGF2α on either Day 5 or Day 7 of the estrous cycle in a 3 × 2 factorial arrangement. Blood samples for progesterone (P4) concentrations were collected at 0, 24, 48, and 72 hours after PGF2α to assess luteolysis (L). Luteolysis was defined on the basis of P4 concentrations at 72 hours using either less than 0.5 ng/mL (L0.5) or less than 1.0 ng/mL (L1.0) as the cut off. Luteolysis was considered “partial” when P4 concentration declined within 24 hours after PGF2α but failed to decline further or, in some cases, increased. Incidence of luteolysis was less (P < 0.01) on Day 5 than Day 7 of the estrous cycle (17.3 vs. 47.6% and 30.4 vs. 77.2%; for L0.5 and L1.0, respectively). Dose of PGF2α increased (P < 0.01) L1.0 (12.5 mg = 38.9%; 25.0 mg = 52.3%; and 50.0 mg = 70.4%). Incidence of partial luteolysis for cows on Day 5 (57.1%) was greater (P < 0.01) than that on Day 7 (19.1%) of the estrous cycle and was more prevalent (P < 0.01) with lower doses of PGF2α (12.5 mg = 49.1%; 25.0 mg = 37.4%; and 50.0 mg = 27.8%). In conclusion, both days of the estrous cycle and doses of PGF2α influenced the incidence of complete and partial luteolysis in Nellore cows and should be an important consideration when devising estrus synchronization programs in this species.