An acylamino phospholipid analogue (2-(R)-N-palmitoylnorleucinol-1-phosphoglycol or (R)-PNPG) was examined for its inhibitory effects against type II phospholipase A2 (PLA2) acting on membranes from Escherichia coli. Using two enzyme sources (rat platelet membranes or recombinant human type II PLA2), (R)-PNPG inhibited phospholipid hydrolysis to a maximal value of 80-85%, half-maximal effect being attained at a substrate/inhibitor molar ratio of 80-250. In contrast, (S)-PNPG was 12-fold less potent and thus provided a control for possible non-specific effects of these polar lipids. However, both analogues exerted only marginal effects on the liberation of [3H]arachidonic acid from rat platelets challenged with calcium ionophore A23187. Since, among various animal species, rat platelets contain by far the highest amounts of this enzyme, our data rule out any possible involvement of secretory PLA2 in arachidonic acid liberation from platelet phospholipids, cytosolic PLA2 appearing in this case as the best candidate able to regulate eicosanoid biosynthesis.