The phosphate carrier of isolated rat liver mitochondria was studied. 2. To investigate the effect of the substrate on the external side of the transport protein, a technique was required by which the extramitochondrial phosphate concentration could be changed without any effect on either the phosphate content or the pH value in the intramitochondrial compartment. This was found when non-respiring mitochondria were incubated with the ionophore nigericin. 3. Using this method variation of the phosphate concentration outside the mitochondria did not protect the essential SH groups located at the external surface of the phosphate carrier. 4. Interrelations within the phosphate carrier unit were investigated in mitochondria in which only one of the two essential thiol groups of the carrier unit was free. These mitochondria were however able to carry out phosphate transport. 5. Alterations of the intramitochondrial pH-induced modification of the position of the single free sulfhydryl group at the external surface of the membrane similarly to the changes which occur in intact mitochondria where both SH groups are free [E. Ligeti and A. Fonyó (1984) Eur. J. Biochem. 139, 279-285]. 6. It is suggested that the sulfhydryl groups essential for the transport function do not participate in the formation of the substrate binding site of the carrier protein. They probably have a role in the conformational change necessary for the translocation step. The possibility of two equivalent but independently functioning parts within the carrier unit is raised.
