A plasma-membrane fraction was isolated from a post-nuclear extract of human neutrophils by centrifugation through a linear sucrose density gradient. This fraction exhibited a Ca2+-dependent adenosine triphosphatase (ATPase) activity that could be differentiated from mitochondrial or myosin ATPase and from plasma-membrane Mg2+-dependent ATPase. When assayed in the presence of [gamma-32P]ATP, the Ca2+-dependent ATPase reaction resulted in the formation of an acid-resistant hydroxylamine-sensitive bond between the gamma-[32P] phosphate group and a membrane protein subunit with an apparent mol.wt. of 135000. Half-maximal activating effect of Ca2+ was found at 82nM and 0.18 microM for the ATPase and the formation of the 32P-membrane complex respectively. Generation of the phosphorylated product attained the steady state at 0 degrees C by about 30s, and was rapidly reversed by ADP. These results suggest that the Ca2+-activated ATPase reaction occurs through the formation of a phosphoprotein intermediate, similar to that described for some Ca2+-dependent ATPase enzymes associated with Ca2+ transport. The possibility thus exists that the neutrophil Ca2+-dependent ATPase catalyses a process of Ca2+ extrusion from the cell, thereby participating in the regulation of several Ca2+-dependent neutrophil functions.