Exoenzyme S (ExoS) is a mono-ADP-ribosyltransferase secreted by the opportunistic pathogen Pseudomonas aeruginosa. ExoS requires a eukaryotic factor, the 14-3-3 protein, for enzymatic activity. Here, two aspects of the activation of the ADP-ribosyltransferase activity of ExoS by 14-3-3 proteins are examined. Initial studies showed that several isoforms of 14-3-3, including β, ξ, η, σ, and ô, activated ExoS with similar efficiency. This implicates a conserved structure in 14-3-3 that contributes to the interaction between 14-3-3 and ExoS. One candidate structure is the conserved amphipathic groove that mediates the 14-3-3/Raf-1 interaction. The next series of experiments examined the role of individual amino acids of the amphipathic groove of 14-3-3ξ in ExoS activation and showed that ExoS activation required the basic residues lining the amphipathic groove of 14-3-3ξ　 without extensive involvement of the hydrophobic residues. Strikingly, mutations of Val-176 of 14-3-3ξ that disrupted its interaction with Raf-1 did not affect the binding and activation of ExoS by 14-3-3. Thus, ExoS selectively employs residues in the Raf-binding groove for its association with 14-3-3 proteins.