In an attempt to define further the sequence of events involved in the activation of rabbit peritoneal neutrophils, we have investigated the effects of trifluperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (compound W7), two compounds with known anti-calmodulin activity, on these cells' ability to respond to several stimuli. Neutrophil granule enzyme release, aggregation and oxygen consumption were monitored. These functions were elicited by the addition of one of the following stimuli to cell suspensions: the chemotactic factor f Met-Leu-Phe, arachidonic acid and the divalent cation ionophore A23187. Aggregation, oxygen consumption and granule enzyme release stimulated by the above three stimuli are all profoundly affected by calmodulin inhibitors. Low concentrations of trifluperazine slightly potentiates aggregation and oxygen consumption. At higher, but below lytic concentrations, compound W7 and trifluperazine significantly depress all three functions examined. In conjunction with previous results and by analogy to studies in other cell types, it therefore appears likely that calmodulin-stimulated events play central roles in the activation of rabbit neutrophils.