Previous studies have shown that platelets exhibit a H2O2 producing, NADH-dependent system that is activated by interaction with particulate material. Current evidence suggests that this system could be critically involved in th generation of chemotactic factor(s). In the present studies, chemotactic activity for polymorphonuclear leukocytes of supernatants derived from zymosan-stimulated human platelets has been evaluated using an agarose gel technique. Supernatants of opsonized zymosan-stimulated platelets showed significant chemotactic activity (migration index = 300 +/- 50), in comparison with supernatants prepared from platelet suspensions stimulated with nonopsonized-zymosan (migration index = 10 +/- 15) or resting platelet supernatants (migration index = 15 +/- 15). Furthermore, a marked increase in chemotactic activity of the opsonized zymosan-treated platelet supernatants was demonstrated after the addition of NADH (migration index = 525 +/- 100). The inclusion of specific inhibitors of the cycloxygenase and lipoxygenase pathways resulted in a marked reduction of chemotactic activity, which was restored in the presence of NADH. Further, the addition of superoxide dismutase completely abolished the chemotactic response induced by NADH. These data suggest that platelets are the source of chemotactic factor(s) derived from the activation of a superoxide generating system.