The role of changes in cytosolic free calcium (Ca) in the activation sequence of the human neutrophil has been monitored by means of the fluorescent probe Quin2. "Complete" secretagogues such as the chemotactic peptide f-Met-Leu-Phe and aggregated IgG, as well as the "incomplete" secretagogue concanavalin A, elicited prompt rises in cytosolic Ca. The rise in cytosolic Ca was in all cases one of the earliest measurable events, consistent with the hypothesis that increments in cytosolic free Ca serve as a signal to activate subsequent physiological responses. The source of cytosolic Ca is principally intracellular, since removal of extracellular Ca had little effect on the rise in cytosolic Ca. Although increments in cytosolic Ca may be essential for neutrophil activation, they are evidently not sufficient; chemotactic levels of f-Met-Leu-Phe elicited optimal increments in cytosolic Ca without triggering degranulation, O-2 generation, and aggregation. Some other factor, associated with high levels of receptor occupancy, must be required for secretion. The tumor promoter phorbol myristate acetate was an exceptional stimulus, since it elicited degranulation, aggregation, and O-2 generation without triggering a rise in cytosolic Ca. Finally, an efflux of Ca is initiated which serves to maintain low intracellular levels of Ca.