In previous work, variation in sperm mobility phenotype was attributed to the proportion of ejaculated fowl sperm containing dysfunctional mitochondria. In the present work, latent mitochondrial dysfunction was inferred from patterns of sperm egress from the oviduct's sperm-storage tubules. In addition, experiments were performed to help explain how mitochondrial function could be compromised in viable sperm cells. Confocal microscopy demonstrated that sperm Ca² content differed between low and high sperm-mobility phenotypes when sperm were stained with rhod-2 AM, a Ca² -specific dye. Fluorescence was associated with the nuclear envelope, a variant of the endoplasmic reticulum, and greater fluorescence was observed in sperm from low sperm-mobility males. Fluorescence was reduced by 50% when motile sperm were rendered immotile by incubation with a Ca² chelator. Thus, a relationship was established between a dynamic intracellular Ca² pool and sperm motility. Sperm N-methy-d-aspartic acid (NMDA) receptors were inferred by the action of d-homocysteinesulfinic acid, a potent NMDA receptor agonist. Seminal plasma from low sperm mobility males was characterized by an elevated glutamate concentration. Thapsigargin, which inhibits the smooth endoplasmic reticulum Ca² pump and thereby promotes Ca² efflux, rendered sperm immotile. This effect was blocked by cyclosporin A, which prevents the formation of the mitochondrial permeability transition pore (PTP) in response to elevated mitochondrial Ca² content. In summary, we propose that 1) glutamate enables Ca² uptake into sperm before ejaculation, 2) excessive Ca² uptake triggers formation of the PTP in a subpopulation of sperm, and 3) sperm mobility is decreased in proportion.