Hyperactivated sperm motility is usually characterized by high-amplitude flagellar bends and asymmetrical flagellar beating. There is evidence that an inositol 1,4,5-trisphosphate (IP₃) receptor-gated Ca² store in the base of the flagellum provides Ca² to initiate hyperactivation; however, the identity of the store was not known. Ca² stores are membrane-bounded organelles, and the only two membrane-bounded organelles found in this region of sperm are the redundant nuclear envelope (RNE) and mitochondria. Transmission electron micrographs revealed two different compartments of RNE, one enriched with nuclear pores and the other containing few pores but extensive membranous structures with enlarged cisternae. Immunolabeling showed that IP₃ receptors and calreticulin are located in the region containing enlarged cisternae. In other cell types, mitochondria adjacent to Ca² stores are actively involved in modulating Ca² signals by taking up Ca² released from stores and also may respond by increasing production of NADH and ATP to support increased energy demand. Nevertheless, bull sperm did not show an increase in NADH when Ca² was released from intracellular stores by thapsigargin to induce hyperactivation. Consistently, no net increase in ATP production was detected when sperm were hyperactivated, although ATP was hydrolyzed at a greater rate. Furthermore, blocking Ca² efflux from mitochondria by CGP-37157, a specific inhibitor of the mitochondrial Na /Ca² exchanger, did not inhibit the development of hyperactivated motility. We concluded that the intracellular Ca² store is the part of RNE that contains enlarged cisternae and that Ca² is released directly to the axoneme to trigger hyperactivated motility without the active participation of mitochondria.