Previous results demonstrating the role of the superoxide anion in human sperm capacitation indirectly suggest that the superoxide anion is not only essential to this process, but is also generated by spermatozoa themselves. Our aim was to provide direct evidence for production of superoxide anion by capacitating spermatozoa. Three chemiluminescence probes were tested to detect the superoxide anion. Luminol- and lucigenin-amplified chemiluminescence of capacitating spermatozoa did not differ from that of background level. However, significant SOD-inhibitable chemiluminescence was observed when the Cypridina luciferin analog, MCLA, was used as probe. The chemiluminescence associated to capacitation of spermatozoa incubated in Ham's F-10 supplemented with fetal cord serum ultrafiltrate was 1270 +/- 77 mV/10 s (with 8 x 10(6) cells/ml; n = 37), and corresponded to levels of sperm hyperactivation (12 +/- 1%) and capacitation (17 +/- 2%) that were significantly different from those of control spermatozoa (4.9 +/- 0.8% and 6 +/- 1%, respectively). MCLA was cell impermeant and superoxide specific. The level of capacitation-associated chemiluminescence was directly related to sperm concentration up to 30 x 10(6) cells/ml. Sperm production of superoxide anion started at the beginning of incubation in capacitating conditions and was sustained over more than 4 h. This is the first direct evidence for the generation, by human spermatozoa, of a sustained level of superoxide anion that is associated with the progressive development of hyperactivation and capacitation.