Two experiments were conducted to assess the repeatability of prolactin responses to a small dose of sulpiride in estrogen-primed geldings in spring and in mares during the estrous cycle in summer. Six long-term geldings each received a single intramuscular injection of 100 mg of estradiol cypionate on March 31, 2011, and were then challenged with an intravenous injection of dl-sulpiride (5 μg/kg of body weight of the racemic mixture) every other day for a total of 8 days. Jugular blood was collected at 0, 10, 20, 40, and 60 minutes after the injection of sulpiride for prolactin measurement. The experiment was repeated with six mares during the summer (July), except that the number of challenges was extended to 15 over 30 days so that any effect of estrous cycle stage could be assessed. Prolactin responses in geldings during April were robust and were varied in a quadratic manner (P < .003) over the eight sulpiride injections, increasing linearly to a plateau by the fourth injection. Mares also displayed robust prolactin responses to sulpiride injections in July, and there was no effect (P > .1) of day of injection and no effect of stage of estrous cycle (follicular phase, early diestrus, or late diestrus). We concluded that prolactin responses to this dose of sulpiride were sufficiently robust and repeatable for use as a paradigm for studies of the relative competitive efficacy and duration of action of various dopaminergic compounds and their vehicular formulations.