This study evaluated the effect of a prostaglandin F2? (PGF) analogue as an ovulatory stimulus in dairy cows and buffaloes raised in the Amazon biome. To this end, three experiments were performed in the state of Rondônia, located in the Amazon biome. In Experiment 1, 22 lactating dairy buffaloes received 2 mg of intramuscular (I.M.) estradiol benzoate (EB) on day 0 and an intravaginal progesterone-releasing device (CIDR) from day 0 to day 9 of the protocol. On days 8 and 9, all cows were given 500 ?g of I.M. d-cloprostenol (PGF analogue). On day 10, buffaloes were divided into two groups to receive 500 ?g of PGF (PGF group, n = 8) or no treatment (CTL group, n = 14), respectively. In Experiment 2, 16 lactating crossbred dairy cows (Holstein x Gir) received 2 mg of EB on day 0 and a CIDR insert from day 0 to day 8. On days 7 and 8, all cows were given 500 ?g of d-cloprostenol. On day 9, cows were divided into two groups to receive 500 ?g of d-cloprostenol (PGF group, n = 8) or no treatment (CTL group, n = 8), respectively. In Experiment 3, 16 lactating crossbred dairy cows (Holstein x Gir) were handled and treated similarly as in Experiment 2, although cows did not receive d-cloprostenol on day 8. Single-point outcome variables were analyzed using one-way analysis of variance (ANOVA), while proportions with dichotomous outcomes were analyzed with the chi-square test. In Experiment 1, there was no difference (P = 0.30) in the ovulation rate between groups, and, on average, 68% of the buffaloes ovulated. Moreover, the treatment did not affect the interval to ovulation (P = 0.61) nor the diameter of the preovulatory follicle (P = 0.47). As for Experiment 2, only one cow, from the PG group, did not ovulate. There were no differences between the CTL and PG groups (P = 0.69) in the moment of ovulation, which occurred in average 82 h after CIDR removal. Finally, in Experiment 3, cows treated with PGF ovulated earlier than those in the CTL group (62.5 ± 5.8 and 94.5 ± 13.5 h, respectively; P = 0.05). Collectively, these results suggest that PGF hastens ovulation in lactating dairy cows, whereas no effect was observed in dairy buffaloes.