Capacitation of spermatozoa is essential for fertilization and is visually characterized by hyperactivated motility. Previous reports have shown that foetal cord serum (FCS) and superoxide anion, O2.-, can trigger human sperm hyperactivation (HA) and capacitation and that superoxide dismutase (SOD) could prevent these processes. We investigated further the role of O2.- and FCS components in human sperm HA and capacitation. Percoll-washed spermatozoa were incubated, at 37 degrees C, in Ham's F-10 medium with 7.5% of FCS, dialyzed FCS (> 12 kD), ultrafiltrate from FCS (FCSu; < 3 kD), or xanthine + xanthine oxidase + catalase (X +XO + cat). Spermatozoa incubated with FCSu were also supplemented with catalase to prevent the loss of motility often observed after 2-3 h of incubation. FCS and dialyzed FCS induced significant levels of HA (10 +/- 1% and 7.7 +/- 0.7%, respectively) that were, however, lower than those observed with FCSu (19 +/- 1%) or X + XO + cat (16 +/- 2%). Similar results were obtained when the lysophosphatidylcholine-induced acrosome reaction (LPC-AR, a measure of sperm capacitation) was evaluated. The presence of SOD in the incubation medium blocked the induction of HA and capacitation by FCS, FCSu, X + XO + cat, as well as the spontaneous HA and capacitation. The enzymatic activity of SOD was needed for the prevention of these processes. Desferrioxamine, up to 100 microM, had no effect on HA and LPC-AR induced by FCSu and X + XO + cat. Addition of SOD to already hyperactivated spermatozoa reversed the HA. These data suggest that spermatozoa need a sustained O2.- generation to maintain HA and proceed to capacitation. We hypothesize that FCSu or the O2.- generated by X + XO + cat activate enzymes, possibly a reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase at the level of sperm membrane.