To further explore the role of the N-terminus of group II phospholipase A2 (PLA2) for enzymatic activity, kinetics of recombinant human synovial fluid PLA2 (rPLA2) and a variant with an extra N-terminal methionine (Met-rPLA2) were analyzed with the substrates [1-14C]oleate-labeled Escherichia coli, vesicular phosphatidylethanolamine, and micellar phosphatidylcholine. While N-terminal variation did not affect assay parameters such as pH-optimum, or optimal concentration of calcium and E. coli substrate, it drastically reduced maximum velocities with all three substrates, without any decrease of substrate affinities, suggesting a role of the N-terminus in the catalytic step(s) subsequent to binding of the substrate. Eleven compounds from various structural classes were found to potently inhibit both enzymes. Several of these compounds were equipotent on rPLA2 and Met-rPLA2; others had significantly lower potency on Met-rPLA2 than on rPLA2. This suggests participation of the N-terminal region of the enzyme in the mechanism of inhibition by the latter compounds. The study provides new evidence for the role of the N-terminus of group II PLA2 for catalytic activity.