Pseudomonas aeruginosa exoenzyme S ADP-ribosylates several GTP-binding proteins of apparent Mr = 23,000-25,000. Exoenzyme S absolutely requires a soluble eukaryotic protein, which we have named FAS (Factor Activating exoenzyme S), in order to ADP-ribosylate all substrates. The rate of ADP-ribosylation of all exoenzyme S substrates increases linearly with time and with the FAS concentration. FAS is wide-spread in eukaryotes but appears to be absent from prokaryotes. We have estimated the molecular mass of the protein to be approximately 29,000 daltons and its pI to be 4.3-4.5. Several bacterial toxins share this sort of requirement for the presence of a eukaryotic protein for enzymic activity. In particular, FAS resembles ADP-ribosylation factor, a 21,000-dalton GTP-binding protein which performs an analogous function for cholera toxin. However, we can find no evidence that FAS binds GTP. In the presence of FAS, exoenzyme S ADP-ribosylates several proteins in lysates of P. aeruginosa. The requirement for a eukaryotic protein for enzymic activity, which is common to several bacterial toxins, may be a device to identify the eukaryotic environment and to ensure that the enzymes cannot function within and harm the toxin-producing bacteria.