Protein-protein interactions play an important role in all cellular processes. The development of two-hybrid systems in yeast and bacteria allows for in vivo assessment of such interactions. Using a recently developed bacterial two-hybrid system, the interactions of the Staphylococcus aureus proteins FemA, FemB and FmhB, members of the FemABX protein family, which is involved in peptidoglycan biosynthesis and beta-lactam resistance of numerous Gram-positive bacteria, were analysed. While FmhB is involved in the addition of glycine 1 of the pentaglycine interpeptide of S. aureus peptidoglycan, FemA and FemB are specific for glycines 2/3 and 4/5, respectively. FemA-FemA, FemA-FemB and FemB-FemB interactions were found, while FmhB exists solely as a monomer. Interactions detected by the bacterial two-hybrid system were confirmed using the glutathione S-transferase-pulldown assay and gel filtration.