We have examined the putative relationship between the presence of neutral pH optimum phospholipase A2 activities and the phospholipid and fatty acid composition in the distinct fractions obtained after subcellular fractionation in a continuous sucrose gradient of human neutrophils. Neutral phospholipase A2 activity was found mainly in the plasma membrane, the azurophilic granules and an unidentified fraction which was a little denser than that containing specific granules and endoplasmic reticulum. These differently located phospholipase A2 activities resulted in activation upon calcium ionophore A23187-cell treatment, but the phospholipase A2 activity found in the unidentified region showed the highest degree of activation after cell treatment with A23187 and hydrolyzed preferentially ethanolamine-containing glycerophospholipids. The subcellular distribution of inositol-containing phosphoglycerides was bimodal, with a main peak at the plasma membrane and another peak at the azurophilic granule region. Choline- and ethanolamine-containing glycerophospholipids showed a broad distribution throughout the gradient, with preponderance in the denser part of the gradient, where the intracellular organelle phospholipase A2 activities were located. Interestingly, ethanolamine-containing glycerophospholipids were shown to be enriched in fractions containing the phospholipase A2 which hydrolyzes preferentially this phospholipid class. Most of the arachidonic acid present in the postnuclear fraction of human neutrophils was found in the plasma membrane and resulted to be the only fatty acid released during A23187 stimulation. Thus, plasma membrane phospholipase A2 seems to be involved mainly in the release of arachidonic acid in A23187-stimulated cells. These findings indicate that differences in substrate specificity found in vitro among the phospholipase A2 activities located in distinct subcellular particulates might be a reflection of the distinct phospholipid and fatty acid composition in these particulates.