The purpose of this study is to investigate how the mitochondrial membrane potentialaffects sperm motility using laser tweezers and a non-ratiometric fluorescent probe,DiOC6(3). A 1064 nm Nd:YVO4 continuous wave laser was used to trap motile sperm at a power of450 mW in the trap spot. Using customized tracking software, the curvilinear velocity (VCL)and the escape force from the laser tweezers were measured. Human (Homo sapiens), dog(Canis lupis familiaris) and drill (Mandrillus leucophaeus) sperm were treated withDiOC6(3) to measure the membrane potential in the mitochondria-rich sperm midpieces. Spermfrom all three species exhibited an increase in fluorescence when treated with theDiOC6(3). When a cyanide inhibitor (CCCP) of aerobic respiration was applied, sperm of all threespecies exhibited a reduction in fluorescence to pre-dye levels. With respect to VCLand escape force, the CCCP had no effect on dog or human sperm, suggestinga major reliance upon anaerobic respiration (glycolysis) for ATP in these twospecies. Based on the preliminary study on drill sperm, CCCP caused a drop in theVCL, suggesting potential reliance on both glycolysis and aerobic respirationfor motility. The results demonstrate that optical trapping in combination withDiOC6(3) is an effective way to study sperm motility and energetics.