Mono-ADP-ribosylation, catalyzed by ADP-ribosyltransferases, is a post-translational modification of proteins in which the ADP-ribose moiety of NAD is transferred to an acceptor protein. Previously, we have identified and cloned a glycosylphosphatidylinositol-linked ADP-ribosyltransferase (Yac-1) from mouse lymphoma cells. Yac-1 enzyme contains three regions (region I,II,III) similar to those found in several bacterial toxins and vertebrate ADP-ribosyltransferases. Site-directed mutagenesis was performed to verify the role of Glu 233 in region III. Mutants E233Q, E233D and E233A were inactive for ADP-ribosyltransferase activity. Thus Glu 233 in Yac-1 is essential for enzyme activity, suggesting that Glu 233 in Glu-rich motif near the carboxy terminus plays a catalytic role in ADP-ribosyltransferase activity.