SUMMARY: The system coupling inorganic phosphate exchange across the osmotic barrier of Micrococcus pyogenes var. aureus (Staphylococcus aureus) strain Duncan is highly specific for phosphate, probably for the H2PO'4 ion. Arsenate, however, can replace phosphate. The exchange is inhibited by thiol reactors, certain anions and certain uncouplers of oxidative phosphorylation. The specificity and kinetics of the exchange suggest that the phosphate is carried in the osmotic barrier as R-phosphate groups moved to and fro across the barrier by thermal agitation, the heat of activation for the movement being 37,400 cal. mole, of which at least 17,700 cal./mole is an entropy component. The exchange of the phosphate of the R-phosphate groups in the barrier with inorganic phosphate on either side may be enzymically catalysed, the free energy of the R-phosphate bond being conserved. Alternatively, the R-phosphate groups may dissociate or hydrolyse, forming free H2PO'4 ion and R, the latter then being unable to return across the barrier until R-phosphate is again formed by the spontaneous uptake of H2PO'4 ion.