Mouse T-cell antigen Rt6.1 is a glycosylphosphatidylinositol ( GPI) -anchored arginine-specific Abp-ribosyltransferase and can transfer ADP-ribose moiety of NAD to an arginine residue of a target protein, forming ADP-ribose-acceptor adducts. Depending on the amounts of ADP-ribose acceptor substrates, a soluble form of Rt6.1 expressed by Escherichia coli can catalyze not only ADP-ribosylation but also NAD glycohydrolysis in vitro. However, it has not yet been determined whether native form of Rt6.1, namely the protein expressed on cell surface as a GPI-anchored form, could catalyze NAD glycohydrolysis. To address this issue, we expressed Rt6.1 on COS-7 cells as a GPI-anchored form and investigated NAD glycohydrolysis by the cells. During incubation with NAD. Rt6.1 CDNA-transfected COS-7 cells hydrolyzed NAD to liberate free ADP-ribose. At the same time, the cells ADP-ribosylated arginine residues of several cell surface proteins. These results indicate that cell surface Rt6.1 catalyzes NAD glycohydrolysis, even in the presence of ADP-ribose acceptor substrate on the cell, and suggest that the NADase activity may also have significant meanings in vivo.