A high performance liquid chromatographic method has been developed for the assay of arginine specific ADP-ribosyl transferases. ADP-ribosylation is an important posttranslational modification which consists of the transfer of an ADP-ribose group from NAD to an acceptor protein. The assay that we developed utilizes commercially available L-arginine methyl ester, LAME, as the acceptor substrate and does not require the use of radiolabels. ADP-ribosylated-LAME is separated from the reaction mixture using a C-8 reverse phase column. Prior to injection, the assay mixture is derivatized with an OPA/2-mercaptoethanol reagent. Fluorescence detection of the OPA derivatized product provides excellent sensitivity and a limit of detection of 300 femtomoles. Total analysis time is 15 minutes with ADP-ribosylated-LAME eluting at 3.9 minutes;Using this assay, we were able to determine the kinetic mechanism of two arginine specific ADP-ribosyl transferases, cholera toxin A subunit and an endogenous transferase from rabbit skeletal muscle. A random sequential mechanism was determined to be the kinetic mechanism for both of the transferases studied. Cholera toxin was reported to have K m's of 5.6 mM and 39 mM for NAD and LAME, respectively. K m values of 0.56 mM and 1.2 mM were determined for NAD and LAME, respectively, using the transferase from rabbit skeletal muscle.