A spectrophotometric assay for the measurement of malondialdehyde and 4 hydroxyalkenals (MA + 4HA) has been evaluated for the detection of sperm pathologies involving oxidative stress. In order to make sensitive measurements of MA + 4HA on human spermatozoa, the stimulation of a lipid peroxidation cascade with a ferrous ion promoter was found to be necessary. The optimal configuration for the promoter was defined (0.64 m M FeSO4 + 20 m M ascorbate for 2 h in Ca2+ and Mg2+ free Hanks' balanced salt solution) and the assay used in a series of studies to elucidate the functional significance of MA + 4HA determinations. Such measurements were found to give highly significant correlations ( p < 0.001) with the loss of motility induced by oxidative stress created either with a xanthine oxidase, free radical generating system or by prolonged incubation under aerobic conditions. Experiments involving the stimulation and suppression of lipid peroxide release from human sperm suspensions, in concert with a bioassay for cytotoxicity, confirmed the strength and causative nature of these associations. Measurements of lipid peroxidation potential in highly purified, leucocyte-free sperm suspensions revealed the presence of inverse correlations with the motility of the spermatozoa, their viability, their competence for sperm-oocyte fusion and, most significantly, the quality of sperm movement in the original semen samples. Similar negative correlations were observed between sperm function and phorbol ester-stimulated reactive oxygen species generation but, unlike the MA + 4HA determinations, these relationships were obfuscated by the presence of leucocytes. We conclude that the measurement of MA + 4HA in human spermatozoa provides important information on the underlying quality of spermatogenesis and should be of value in the clinical diagnosis of infertility involving oxidative stress and the selection of patients for antioxidant therapy.