The possible involvement of membrane-bound calcium in the mechanism of action of leukotriene B4 was examined using the fluorescent chelate probe, chlortetracycline. Leukotriene B4 was found to cause a rapid release of membrane-bound calcium at physiologically relevant concentrations. This effect of leukotriene B4 is stereospecific and its magnitude is decreased upon the transformation of leukotriene B4 into its omega-hydroxy and omega-carboxy metabolites. The pool of calcium affected by leukotriene B4 appears to be the same as that released by other chemotactic factors such as formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe). Similarly, preincubation with f-Met-Leu-Phe results in a decreased responsiveness of the cells to the addition of leukotriene B4. These results extend further the analogy between the mechanism of action of peptidic and lipid chemotactic factors, and emphasize the central role of the intracellular redistribution of calcium, as inferred and monitored by chlortetracycline fluorescence and steady-state isotopic flux studies, in neutrophil activation.