A micellar electrokinetic chromatographic method that allows simultaneous determination of both nucleotidase and transferase activities of cytosolic 5'-nucleotidase III is presented. This electrophoretic approach was successfully applied to human erythrocyte lysates to monitor the enzyme activities indicated above, using either physiological substrate or the nucleoside analogue 3'-azido-3'-deoxythymidine as the phosphate acceptor.