Alligators are territorial animals that exhibit interspecies aggression which often results in serious injuries. However, despite the fact that these animals live in marsh environments that harbor a wide variety of potentially infectious microbes, these wounds heal rapidly and generally without infection. Leukocytes in higher eukaryotes, such as mammals, migrate to the site of an infection and produce superoxide ions (O2-) to help fight infection. This study was conducted to determine if alligator leukocytes were capable of O2- production. We used WST-1, a tetrazolium salt which can be reduced to a formazan compound with high molar absorptivity at 438 nm, to probe the production of O2- by alligator leukocytes. Incubation of alligator whole blood with WST-1 resulted in a time- and concentration-dependent increase in absorbance of the plasma at 438 nm. The reduction of WST-1 was inhibited in a concentration-dependent manner by superoxide dismutase, an enzyme that catalyzes the reduction of O2- to H2O2, confirming that the reduction of WST-1 was due to the presence of O2-. It is interesting that the production of O2- by the alligator leukocytes required no external stimulation while human leukocytes must be stimulated with an immunological challenge before producing O2-. Incubation of alligator blood with nitroblue tetrazolium, a compound that can be reduced to an insoluble purple diformazan, resulted in no staining of alligator leukocytes. Examination of leukocytes by confocal light microscopy showed clustered concentrations of cytoplasmic granules near the plasma membrane, confirming that the O2- is translocated outside the cells by degranulation.