Cisplatin [cis-Diamminedichloroplatinum (II), DDP] generated superoxide anion (O2-), as measured by chemiluminescence from a Cypridina luciferin analog (CLA), by interaction with calf thymus DNA (ct-DNA) in phosphate-buffered saline (PBS) at pH 7.6. The intensity of chemiluminescence of CLA (CCLA) increased with increased concentration of DDP, ct-DNA or CLA in the limited range of concentrations of these agents tested. DDP-induced CCLA was completely inhibited by the addition of an O2- scavenger, superoxide dismutase (SOD), or an antioxidant, ascorbic acid. Heat-inactivated SOD did not affect CCLA. The hydroxyl radical (OH.) scavengers, mannitol, ethanol, and formic acid, also caused a partial suppression of CCLA, suggesting that DDP produced OH. as well as O2- in the cell-free system used.