A novel approach based on de and ac cyclic voltammetric techniques to evaluate interfacial enzymatic activity is presented. As an illustrative example, the hydrolytic effect of phospholipase A(2) (PLA(2)) on an L-a-dioleoylphosphatidylcholine (DOPC) monolayer adsorbed onto a mercury electrode surface has been studied and kinetic parameters have been evaluated. The enzymatic activity obtained in this study is of the order of 10(-1) min(-1). A dramatic effect of [Ca2+] on the hydrolysis of DOPC monolayers by PLA(2) has been observed where the hydrolytic rate constant exhibits a peak-shaped dependence on [Ca2+] with a maximum rate at a Ca2+ concentration of about 6 mM. Ca2+ concentrations above 6 mM appear to have an inhibitory effect on the hydrolysis. Control studies with the nonhydrolyzable substrate analog, N-oleoyl-D-sphingomyelin (SM), demonstrate that the PLA(2) does not simply displace the intact phospholipid molecules from the electrode surface. Thus, the observed electrochemical response arising from PLA(2) is due solely to those enzyme (PLA(2)) molecules that are adsorbed onto the mercury electrode surface following the displacement of the hydrolytic products (fatty acids and lysophospholipids). It has also been found that Ca2+ has virtually no effect on the adsorption dynamics of PLA(2) onto a mercury electrode surface, with the adsorption rate constant being of the order of 10(6) min(-1) over a Ca2+ concentration range of ca. 0-10 mM. A comparison of the chemical reaction (hydrolysis) rate constant (10(-1) min(-1)) with the adsorption rate constant (10(6) min(-1)) suggests that the rate-limiting step of the enzymatic reaction is the chemical step itself rather than the binding step of the enzyme from the bulk solution to the interface, consistent with other reports in the literature.