An electrochemical method was used to probe the interaction of actinomycin D (AMD) and calf thymus DNA (ctDNA) in phosphate buffer solution (pH 7.0) at an oxidized waxed graphite electrode. AMD showed a pair of nonsymmetric peaks at -0.51 and 0.19 V in cyclic voltammetry. As a result of reaction with ctDNA, the voltammetric peaks of AMD disappeared. The diffusion coefficient of free AMD (D-f = 1.9 x 10(-5) cm(2) s(-1)), binding constant (K = 7.54 x 10(9) cm(3) mol(-1)) and binding site size (s = 8.1) of the AMD-DNA complex were obtained simultaneously by nonlinear fit analysis of voltammetric data based on an electrochemical equation, which was derived to examine the interaction of DNA and irreversibly electroactive compounds. The results demonstrate that AMD binds tightly to ctDNA. The AMD phenoxazone ring intercalates between the DNA base pairs. The two side chains of the depsipeptide extend along the minor groove of DNA and cover eight base pairs. The study provides a convenient and sensitive approach to estimate affinity parameters of the interaction between DNA and targeting compounds. (C) 2003 Elsevier Science B.V. All rights reserved.