AbstractOptimum conditions were established for the generation and measurement of luminoldependent chemiluminescence (CL) in human polymorphonuclear leucocytes (PMNL) stimulated with a variety of particulate and soluble agents. Several factors had a particular influence on the kinetics of CL stimulated by the chemotactic peptideN‐formyl‐L‐methionyl‐L‐leucyl‐L‐phenylalanine (fMLP). Two peaks, both azide‐sensitive, were observed at 21°C and 25°C. but these increased in magnitude and merged t o give a single, early peak when the temperature was increased t o 37°C. Pre‐exposure of PMNL to a buffer containing calcium was essential for the expression of both phases of fMLP‐stimulated CL, while the second peak decreased dramatically if the cells were stored at 4°C for 4 hours before assay. In contrast, storage of PMNL at 4°C for up t o 8 hours in a buffer without divalent cations did not alter the kinetics or magnitude of CL induced by other stimuli, and had the benefit of minimizing the rate of cell aggregation. This study confirms that measurement of luminol‐dependent CL in stimulated PMNL is a useful analytical tool, but shows that careful attention t o experimental design is required t o ensure that the observed CL provides a true measure of the par