Clostridium sordelliilethal toxin (LT), a cytotoxin which causes preferential destruction of the actin cytoskeleton, has been recently identified as glucosyltransferase to modify the low molecular mass GTPases Rac, Ras and Rap. We report here on LT produced byC. sordelliistrain 6018 which glucosylates in addition to Rac, Ras and Rap the Ral protein. LT from strain VPI9048 however does not glucosylate Ral. Besides recombinant Ral, cellular Ral is also substrate. In the GDP-bound form, Ral is a superior substrate to the GTP form. Acceptor amino acid for glucose is threonine-46 which is equivalent to threonine-35 in H-Ras located in the effector region. The Ral-glucosylating toxin is a novel isoform of Ras-modifying clostridial cytotoxins.