The relationship between lipid peroxidation and the functional competence of human spermatozoa has been investigated in a cohort of 31 infertility patients. Lipid peroxidation was assessed using a sensitive fluorometric assay for the generation of malondialdehyde in response to the presence of a ferrous ion promoter. Sperm function was evaluated by monitoring the movement characteristics of these cells and their capacity for sperm–oocyte fusion. Each sample was separated into high- and low-density sperm populations on discontinuous, two-step (40%:80%), Percoll gradients prior to analysis. The way in which individual ejaculates fractionated on these gradients was highly positively correlated (P < 0.001) with the lipoperoxidation status of the spermatozoa; the greater the potential for malondialdehyde generation, the higher the proportion of cells entering the low density region of the gradients. The lipoperoxidation potential of the freshly prepared spermatozoa was also highly predictive (P = 0.0001) of their capacity for movement at 3 and 24 h and their ability to exhibit sperm–oocyte fusion in response to the ionophore A23187. The potential for malondialdehyde generation in the 40% and 80% Percoll fractions was positively associated with midpiece abnormalities in the spermatozoa. These results emphasize the importance of lipid peroxidation in the pathophysiology of male infertility and suggest a mechanism by which such damage might arise.