Objective: To investigate sperm mitochondrial integrity through analysis of mitochondrial membrane potential (Δψ) and to correlate the energy status with variations in sperm motility. Design: Experimental study. Setting: Seminology Laboratory, University of Rome, Italy. Patient(s): Two hundred thirteen semen samples from the same number of patients, divided into two groups on the basis of their motility: group A, 185 samples with linear motility and group B, 28 samples with nonlinear motility. Intervention(s): Evaluation of sperm motility. Main Outcome Measure(s): Sperm mitochondrial integrity evaluated with a fluorimetric method using the cationic lipophilic stain JC-1. Result(s): The mean FL2 (percentage of sperm with high and low ψ) for group A was 46.19 ± 23.25 and for group B, it was 48.32 ± 24.43. There was no significant difference between the groups. There was a positive correlation between both FL2 and linear motility and FL2 and sperm vitality in group A; both correlations were statistically significant. In group B, there was a positive correlation between FL2 and nonlinear motility and FL2 and sperm vitality; again, both correlations were statistically significant. Conclusion(s): Our data reveal a positive correlation between total motility and Δψ, suggesting that sperm motility may be dependent on the functional integrity of the mitochondria. © 2011 by American Society for Reproductive Medicine.