The concentration of cytosolic ionized calcium, [Ca2+]i, was measured in intact neutrophils by use of a fluorescent indicator trapped in the icytoplasm. A given rise of [Ca2+]i elicited by the chemotactic peptide formylmethionylleucylphenylalanine (FMLP) was associated with a much greater degree of superoxide generation and myeloperoxidase secretion than was the same or larger [Ca2+]i produced by a specific calcium ionophore, ionomycin, which bypasses cell surface receptors. Thus, FMLP appears to generate some important excitatory signal in addition to a rise in [Ca2+]i and exocytosis and superoxide generation in neutrophils may not be simply dependent on [Ca2+]i as is widely supposed.