When polymorphonuclear leukocytes (PMNL) interact with the soluble stimuli FMLP or PMA, the cells increase their production of oxidative metabolites. This increased production can be measured as luminol amplified light emission or chemiluminescence (CL). The chemiluminescence of human PMNL has been investigated, and it was found that the chemoattractant FMLP induced a bimodal response with a sharp peak of activity within 1 min, and a second peak after around 5 min. In contrast, PMA induced a one peak response reaching a maximum around 15 min after stimulis addition. Despite the fact that strictly standarized conditions for cell preparation and CL measurements were used, an extensive variability, especially in the response to FMLP was observed. Dismutation of O(2) by the addition of superoxide dismutase (SOD) or consumption of H2O2 by addition of catalase resulted in very small reductions of the CL compared to the effects on cytochrome c reduction and scopoletine fluorescence, respectively. Furthermore, since SOD reduced also the CL generated from a cell-free peroxide-peroxidase system, the specificity of SOD in the CL reaction could be questioned. Expression of CL and the effects of SOD and catalase was furthermore found to be dependent on the number of responding cells. Analysis of the effects of SOD and catalase on the bimodal FMLP response show that the first peak is strongly inhibited, whereas a very small effect upon the expression of the second peak is obtained. These results indicate, that since SOD and catalase are expected to reduce only extracellularly generated oxidative metabolites, the first peak of the FMLP response is of extracellular origin, whereas the second peak and most of the PMA induced response are cell associated or intracellular phenomena.