Mono-ADP-ribosylation is a posttranslational modification of proteins in which the ADP-ribose moiety of NAD is transferred to proteins and is responsible for the toxicity of some bacterial toxins (e.g., cholera toxin, pertussis toxin). Five members of the mammalian NAD:arginine ADP-ribosyltransferases (ART1-5) were cloned from various tissues. The ART1 proteins from cardiac and skeletal muscle and lymphocytes are glycosylphosphatidylinositol (GPI)-anchored proteins that modify extracellular adhesion molecules. A second ADP-ribosyltransferase (ART5) was cloned from mouse lymphoma cells, but is expressed predominantly in testis. The ART5 protein is membrane-bound but, unlike the ART1 enzyme, appears not to be GPI-anchored. The ART1 and ART5 enzymes, expressed as glutathione-S-transferase fusion proteins in E. coli, were used to compare their ADP-ribosyltransferase and NAD glycohydrolase activities. Using agmatine as the ADP-ribose acceptor, ART1 was predominantly an ADP-ribosyltransferase, whereas the transferase and NAD glycohydrolase activities of recombinant ART5 were equivalent. The deduced amino acid sequence of the mammalian ART proteins contains three consensus regions common to several bacterial toxin transferases. These regions have been shown to form, in part, the active site of the bacterial toxin transferases. Site-directed mutagenesis of the proposed active-site glutamate of ART1 abolished transferase activity. Further, when the carboxy-terminal half of ART1, which contains the regions postulated to be crucial to maintaining a functional catalytic site, was expressed as a GST-fusion protein, the truncated enzyme retained NAD glycohydrolase activity consistent with the hypothesis that there is a common mechanism of NAD binding and catalysis among ADP-ribosyltransferases.