The 8-anilinonaphthalene-1-sulfonate (ANS) fluorescence intensity of ANS-Naja naja atra (Taiwan cobra) phospholipase A2 (PLA2) complex increased with the addition of Ca2+, but the observed fluorescence enhancement markedly decreased after methylation of His-47 in PLA2 molecule. However, the binding affinities of methylated PLA2 for ANS and Ca2+ were similar to or even greater than those observed with native PLA2. These results, together with the finding that ANS electrostatically interacted with His-47 of PLA2, suggest that the increase in the intensity of ANS fluorescence upon the addition of Ca2+, in part, arises from the ionic interaction of His-47 with ANS being perturbed by the binding of Ca2+.