A ribosyltransferase from C. botulinum type D ADP-ribosylated a protein of 22 kDa (p22) in human astrocytoma (1321N1) cells. ADP-ribosylation of membrane-bound p22 was potentiated by 2 mM MgCl2 or guanine nucleotides but was much reduced in the presence of 10 mM Mg2+ plus GTP gamma S. p22 was immunoprecipitated by a monoclonal antibody (142-24E05) raised against a peptide sequence common to the ras gene family but not by other ras or G-protein antibodies. p22 was also ADP-ribosylated in Drosophila but was not detected in Dictyostelium. These data suggest that the 22 kDa botulinum toxin substrate is a GTP-binding protein and a member of the ras protein family.