Ejaculated mammalian sperm become fertilization-competent after undergoing physiological changes in the female?s reproductive tract, namely capacitation and hyperactivation. The latter is characterized by a change in sperm motility from symmetrical flagellar bends to vigorous, asymmetrical, high-amplitude flagellar bends, with the pattern of motility being species-specific. The main trigger responsible for the initiation of hyperactivated motility is an increase in intracytoplasmic Ca2+ arising potentially from both intracellular and/or extracellular sources. Therefore, pharmacological reagents that increase intracytoplasmic Ca2+ have been used to induce hyperactivation in sperm from a variety of species. Recently, hyperactivation has been characterized for stallion sperm using procaine treatment, a reagent hypothesized to increase membrane permeability to extracellular Ca2+. Since hyperactivation of stallion sperm is crucial for supporting in vitro fertilization (IVF), this study examined the effect of exposing equine sperm to different concentrations of caffeine, thimerosal and thapsigargin, three reagents demonstrated to induce hyperactivation in other species. Sperm from three ejaculates from each of three stallions were exposed to concentrations gradients of each of these reagents to examine their effect of sperm hyperactivation using a computerized sperm motility analyzer (CASA) to measure motion parameters associated with this pattern of motility. In all experiments, positive controls included treatment with procaine. All three reagents tested failed to induce hyperactivation in the stallion, suggesting physiological differences in the response of stallion sperm to these reagents as compared to other species.