Two experiments were conducted to assess the timing and synchrony of ovulation, plasma LH concentrations, and pregnancy rate in Murrah buffaloes (Bubalus bubalis) treated with the Ovsynch (GnRH-PGF 2α -GnRH) protocol. In Experiment 1, 10 non-lactating cycling buffaloes received 10μg of a GnRH analogue i.m. (buserelin acetate) without regard to the stage of the estrous cycle (day of treatment, day 0), followed by 25mg of PGF 2α i.m. (dinoprost thromethamine) 7 days later. A second-treatment of the same GnRH analogue (10μg, i.m.) was given 48h after PGF 2α . Ovulation was confirmed by transrectal palpation (at 2-h intervals) from the second-GnRH treatment to detection of ovulation or up to 96h after the second-GnRH treatment. Plasma LH concentrations were determined in blood samples collected at 15-min intervals for 6h, starting at the second-GnRH treatment, and thereafter at 2-h intervals until 2h after detection of ovulation. Ovulation occurred in 9/10 buffalo (90%) 23.3±1.3h (mean±S.E.M.; range 20–32h) after the second-GnRH treatment. Peak LH concentrations 13.5±3.5ng/mL (range 3.9–40.0ng/mL) occurred 2.1±0.1h (range 1.2–3.0h) after the second-GnRH treatment. In Experiment 2, 15 lactating, cycling buffaloes were subjected to the Ovsynch protocol, with fixed-time AI 12 and 24h after the second-GnRH treatment and 75 lactating buffaloes were inseminated, approximately 12h after detection of spontaneous estrus. Pregnancy rates were 33.3% for TAI and were 30.7% for buffaloes inseminated following spontaneous estrus (P=0.84). In conclusion, the Ovsynch protocol effectively synchronized ovulation in Murrah buffaloes and resulted in conception rates (to two fixed-time inseminations) that were comparable to those achieved with a single AI after detection of spontaneous estrus.