The accumulation of mitochondrial DNA (mtDNA) mutations has been suggested to be an important contributor to human aging and degenerative diseases. In previous studies, we found an age-dependent increase of mtDNA mutations in various human tissues. Sperm motility is one of the determinants of male fertility. The possible relationship between mtDNA deletions and diminished fertility and motility of sperm was explored in the present study. We examined accumulation of the 4977-bp mtDNA deletion in spermatozoa obtained from patients with infertility or subfertility and compared these values with those of normal individuals. Using polymerase chain reaction (PCR) techniques, we determined the frequency of occurrence and the proportion of mtDNA with the 4977-bp deletion in human spermatozoa with different motilities. Human spermatozoa were separated by self-migration in Percoll gradients into five fractions with different motility scores. The highest frequency of occurrence of the 4977-bp mtDNA deletion was found in sperm in the fraction with the lowest motility. The results revealed a negative correlation between sperm motility and the proportion of 4977-bp-deleted mtDNA. Furthermore, we found a significantly higher incidence of the 4977-bp mtDNA mutation in patients with asthenospermia, oligospermia, and primary infertility compared to normal individuals. These findings suggest that mtDNA mutations may play an important role in some pathophysiological conditions in human spermatozoa.