2-Acylamino-alkyl phospholipids are potent competitive inhibitors of 14-kDa phospholipases A2 (e.g., human nonpancreatic secretory PLA2). As concluded from X-ray studies the amide hydrogen of these inhibitors forms a hydrogen bond to His-48 in the active site of the enzyme. We investigated the quantitative contribution of this hydrogen bond to inhibition using especially designed inhibitors that bear different acyl chains with and without electron withdrawing or donating substituents, thus differing in amide acidity. Relative free enthalpies DeltaDeltaG of enzyme-inhibitor complex formations were calculated from Xi(50) values determined by pH-stat titration using a mixed micelles assay and PLA2 from Naja mocambique mocambique. A quantitative relationship between amide acidity and DeltaDeltaG values is presented. Comparison of isoacidic and isosteric inhibitors reveals that (i) the hydrogen bond of the amide proton to His-48 is crucial for strong PLA2 inhibition, (ii) regardless of the headgroup unsubstituted N-acyl groups result in optimal amide acidity for PLA2 inhibition and (iii) the exceptionally strong inhibition by acetamides and the isosteric fluoroacetamides is due to an additional steric effect.