Collective sperm motility is characterized in terms of periodic aggregation or cooperation among cells, which are seen under a microscope as a continuous wave motion. In this study, sperm motility that was made dependent on mitochondrial activity (glycolysis inhibited) was significantly inhibited by mM exogenous calcium while glycolytic-dependent (mitochondrial respiration inhibited) motility was not inhibited under these conditions. At intracellular Ca2+ above 400 nM, sperm motility was inhibited independently of the source of ATP. At [Ca2+]i of approximately 110 nM, mitochondrial- or glycomitochondrial-dependent motility showed 75 or 0% inhibition, respectively, indicating higher sensitivity of mitochondrial-dependent motility to [Ca2+]i in comparison to glycolytic-dependent motility. Under the latter conditions, intracellular level of ATP was reduced by 16% only, indicating that the 75% inhibition of mitochondrial-dependent motility was not due to reduction in ATP. The inhibition of mitochondrial-dependent motility by mM extracellular Ca2+ can be prevented by incubating the cells at pH 6.5 instead of 7.6. At pH 7.6, calcium probably interacts with negative sites on the cell surface and interferes in cell-to-cell interactions, which are important to achieve collective motility. At more acidic pH (6.5) these negative sites are probably protonated; thus Ca2+ cannot interact with them and no inhibition of motility could be seen. Glycolytic-dependent motility was not inhibited by extracellular Ca2+, since the pH of the medium becomes acidic under these conditions. Like Ca2+, mersalyl, which interacts with Ca2+ binding sites on the cell surface, showed significant inhibition of mitochondrial-dependent motility without any effect on glycolytic-dependent motility. Collective motility was directly correlated with fertility. These data are significant for establishing better conditions for spermatozoa treatment when artificial insemination or in vitro fertilization is concerned.