In the presence of extracellular calcium and magnesium, a series of chemotactic oligopeptides and C5a caused aggregation of human polymorphonuclear neutrophils (PMNs). This cellular response developed rapidly and began to reverse 2 min after exposure to the chemotactin. In the absence of the bivalent cations, none of the chemotactins stimulated the aggregation response. If cells were first exposed to a chemotactin and then treated with calcium and magnesium, aggregation was detected only after addition of the cations, and the magnitude of the response fell sharply as the interval between the addition of chemotactin and addition of cations was lengthened: when this interval exceeded 2 min, aggregation was barely detectable. This loss of reactivity persisted even when cells were re-exposed to fresh chemotactic factor and washed between the first and second exposures. In all instances, however, loss of cellular reactivity was highly selective: cells preincubated with any chemotactic oligopeptide were hyporesponsive to subsequent stimulation with an oligopeptide but remained fully responsive to C5a; cells preincubated with C5A were hyporesponsive to C5a but retained their responsitivity to the oligopeptides. Because this selectivity parallels the known specificities of these chemotactic factors for their receptors in or on the neutrophil, desensitization may reflect functional loss of receptors after stimulation. Alternatively, this selectivity may indicate that morphologically identical neutrophils contain subpopulations of cells with varying reactivities to receptor-bound chemotactic factors. In either event, desensitization may be useful in functionally defining chemotactic factors and their respective receptors. The rapidity of development of desensitization suggests that it may operate to limit or moderate various in vitro and in vivo neutrophil responses to chemotactic factors.