We have presented here the biochemical analysis of the E. faecalis gluconate PTS component EIIA and of its complex with EIIB. We show that EIIAgnt is active in vitro and demonstrate by mutagenesis and mass spectrometry analysis that His-9 is its phosphoryl carrying residue during these reactions, which is also in perfect agreement with structural data [18]. Analysis of the EIIAgnt–EIIBgnt interaction by ITC, AUC, native PAGE and chemical crosslinking experiments revealed a strong interaction and a 2:2 stoichiometry of the complex. Also considering that both ONX-0914 in vivo are most likely present in equimolar amounts due to their gene organization in one operon, our data suggest that the 2:2 EIIAgnt:EIIBgnt complex is of physiological relevance. Furthermore, the observed destabilization of the complex upon phosphorylation of EIIBgnt seems to ensure the rapid accessibility of the phosphoryl group for the subsequent transfer to the target sugar. Compared to the short-lived P~EIIA/EIIB complex of the E. coli chitobiose PTS [27] on the one hand and the covalently linked EIIAman and EIIBman domains of the mannose PTS on the other hand, the stable EIIAgnt:EIIBgnt complex might represent an evolutionary intermediate between a system of transiently interacting, single components and multidomain proteins.