Although "secretory" type II 14-kDa phospholipase A2 (sPLA2) activity has been described in neutrophils, direct evidence of enzyme secretion has been elusive. We have used immunogold electron microscopy with polyclonal and monoclonal antibodies to sPLA2 to demonstrate localization of the enzyme to granules of resting human neutrophils and translocation to phagolysosomes. Soluble stimuli such as calcium ionophore A23187 stimulate loss of cell-associated enzymatic activity. Supernatant fluids from stimulated neutrophils lack measurable PLA2 but contain proteases which inactivate exogenous sPLA2. The use of alpha-1-antitrypsin as a protease inhibitor permitted this first demonstration of secretion of PLA2 activity from stimulated human neutrophils.