A relationship between extracellular Ca ², fowl sperm phospholipase A₂ activity, long-chain acylcarnitine content, and motility was demonstrated in previous work. Sperm motility appeared to depend upon Na -dependent Ca ² cycling when sperm were incubated at body temperature without glucose. In the present work, motility decreased as a function of time when sperm were incubated in 2 mM Ca ² prepared with either buffered isotonic sucrose or LiCl. However, this effect was less pronounced in the case of LiCl. The sparing effect of Li was attributed to the mitochondrial Na /Ca ² exchanger. Motile concentration decreased exponentially in response to micromolar concentrations of CGP 37157, a specific inhibitor of the mitochondrial Na /Ca ² exchanger. KB-R7943 mesylate, an inhibitor of the reverse mode of the Na /Ca ² exchanger, prevented re-initiation of motility when exogenous Ca ² was added to sperm rendered immotile by incubation with 1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, a high-affinity Ca ² chelator. The presence of voltage-gated Ca ² channels was confirmed by the effect of nifedipine on motile concentration. Neither motile concentration nor straight line velocity was affected by either ouabain or orthovanadate, which inhibit Na -K ATPase and Ca ²-ATPase, respectively. In summary, we infer that 1) fowl sperm motility is dependent upon extracellular Ca ² cycling through mitochondria; 2) such cycling is dependent upon extracellular Na ; and 3) fowl sperm conserve ATP by moving neither Na nor Ca ² by active transport. Understanding the relationship between mitochondrial Ca ² cycling and ATP production may be applicable to long-term semen storage.