The effects of pathologically relevant concentrations (0.38 to 12.5 µM) of the proinflammatory, Pseudomonas aeruginosa-derived pigment 1-hydroxyphenazine (1-hp) on Ca²⁺ metabolism and intracellular cyclic AMP (cAMP) in N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 1 µM)-activated human neutrophils, as well as on the release of myeloperoxidase (MPO) and elastase from these cells, have been investigated in vitro. Ca²⁺ fluxes were measured by the combination of a fura-2/AM-based spectrofluorimetric method and radiometric procedures, which together enable distinction between net efflux and influx of the cation, while radioimmunoassay and colorimetric methods were used to measure cAMP and granule enzymes, respectively. Coincubation of neutrophils with 1-hp did not affect intracellular cAMP levels or the FMLP-activated release of Ca²⁺ from intracellular stores but did retard the subsequent decline in the chemoattractant-induced increase in the concentration of cytosolic free Ca²⁺. These effects of 1-hp on the clearance of Ca²⁺ from the cytosol of activated neutrophils were associated with decreased efflux of the cation from the cells and increased release of MPO and elastase, while the delayed store-operated influx of the cation into the cells was unaffected by the pigment. The plasma membrane Ca²⁺-ATPase rather than a Na⁺-Ca²⁺ exchanger appeared to be the primary target of 1-hp. These observations suggest that the proinflammatory interactions of 1-hp with activated human neutrophils are a consequence of interference with the efflux of cytosolic Ca²⁺ from these cells.