Exposure of human neutrophils to micromolar concentrations of both hydrolyzable and nonhydrolyzable purine nucleotides caused the generation of transient rises in intracellular calcium (Ca2+), Ca2+ fluxes across the membrane, and primed the cells for enhanced production of superoxide (O2-) when subsequently exposed to agonists such as FMLP and arachidonic acid. The neutrophils were most sensitive to adenosine triphosphate (ATP) and ATP-gamma-S, which produced Ca2+ transients and enhanced O2- production at concentrations as low as 1 to 5 mumol/L, with a doubling of O2- generation at 25 to 50 mumol/L. Adenosine diphosphate (ADP), guanosine triphosphate (GTP), and 5′- adenylylimidodiphosphate (AMP-PNP) required approximately 10-fold higher concentrations to cause similar effects. Adenosine did not cause Ca2+ fluxes or a Ca2+ transient and was inhibitory of O2- production. There was a strong correlation between a nucleotide's ability to generate a Ca2+ response and its ability to enhance O2- generation. Nitrogen cavitation and subcellular fractionation of the neutrophils after a brief exposure to ATP, ATP-gamma-S, and AMP-PNP revealed that the enhanced O2- generating capacity was stable and detectable in a cell-free assay system. By combining variously treated cytosolic and membrane fractions, it was found that the enhanced O2- production was attributable to a modification of a component(s) of the cytosol.