The cell activation inhibitor CI-959 [5-methoxy-3-(1-methylethoxy)-N-1H-tetrazol-5-ylbenzo[ b]thiophene-2- carboxamide, monosodium salt] was evaluated for its effects on human neutrophil functions. CI-959 inhibited spontaneous migration and chemotaxis toward N-formyl-methionyl-L-leucyl-L-phenylalanine (fMLP) with 50% inhibition (IC50) values of 3.6 and 3.1 microM, respectively. CI-959 also inhibited superoxide anion generation in response to C5a, fMLP, serum-opsonized zymosan (SOZ), concanavalin A (Con A), and calcium ionophore A23187 with IC50 values of 2.5, 4.7, 14.5, 5.4, and 14.8 microM, respectively. In comparison, CI-959 inhibited myeloperoxidase microM, respectively. In comparison, CI-959 inhibited myeloperoxidase release in response to C5a, fMLP, SOZ, and Con A with IC50 values of 11.6, 16.1, 7.5, and < 1.0 microM, respectively, while inhibiting the response to A23187 by only 5.5% at 100 microM. At concentrations up to 100 microM, CI-959 had no effect on the respiratory burst or degranulation in response to L-alpha-1,2-dioctanoylglycerol (DiC8) or phorbol 12-myristate 13-acetate (PMA). In addition, the compound inhibited leukotriene B4 release stimulated by fMLP and SOZ (IC50 values 4.0 and 2.5 microM, respectively), while having less activity against the A23187-stimulated response (IC50 > 100 microM). These results demonstrate that CI-959 inhibits cellular responses to stimuli that mobilize intracellular calcium. For cellular responses to inophore-mediated calcium influx, only oxygen radical production was inhibited by CI-959. CI-959 was further evaluated for its effects on neutrophil stimulus-response coupling. At 100 microM, CI-959 had no effect on human neutrophil phospholipase C or protein kinase C. CI-959 inhibited fMLP-stimulated intracellular calcium mobilization and calcium influx with IC50 values of 16.7 and 3.1 microM, respectively, and exhibited less potent calmodulin antagonist activity (IC50 = 90.5 microM). These results indicate that CI-959 may exert its stimulus- and response-specific inhibitory effects on neutrophil functions, in part, through inhibition of calcium-regulated signalling mechanisms.