The objective of this study was to characterize the effect of dose and type of cloprostenol (CLO) on the luteolytic response of dairy cattle during the Ovsynch protocol under different oestrus cycle and physiological characteristics. Twelve non-lactating dairy cows and 111 lactating dairy cows were used in three experiments. In Experiment I, cows were synchronized so that they had only a 5.5- to 6-day-old corpus luteum (CL) at the time of the prostaglandin F2α (PGF2α) treatment of Ovsynch. In Experiment II, cows were synchronized so that they had at least a CL of approximately 14 days old at the time of PGF2α treatment and an accessory CL if they had responded to the first GnRH of Ovsynch. Furthermore, in each experiment, cows received either a standard or a double dose of d-CLO as the luteolytic treatment. In Experiment III, lactating cows were blocked by parity and assigned to one of three luteolytic treatments during Ovsynch: 500 μg d,l-CLO, 150 or 300 μg of d-CLO. In Experiment I, the dose of d-CLO had an effect (p = 0.08) on the percentage of cows with full luteolysis, but not in Experiment II (p > 0.1). More cows in Experiment II had full luteolysis than did cows of Experiment I (87% vs 58%, respectively; p = 0.007). In Experiment III, 87.1%, 84.4% and 86.2% lactating dairy cows had full luteolysis and 37.8%, 36.8% and 36.1% of cows became pregnant after treatment with 500 μg d,l-CLO, 150 or 300 μg of d-CLO, respectively (p > 0.05).