The phosphate carrier from bovine heart mitochondria was reconstituted into liposomes by the removal of detergent using hydrophobic ion-exchange columns. Reversible blocking of the carrier function during chromatographic steps was possible by the application of the inhibitor p-(chloromercuri)benzenesulfonate at low temperature. Thus, both forward and backward exchange experiments for kinetic characterization of Pi/Pi-antiport as well as the Pi/H(+)-symport could be performed. The maximum rate of Pi/Pi-antiport was 90 mumol min-1 (mg protein)-1. Only one single half-saturation constant (Km) for phosphate was observed at each side of the membrane under antiport conditions, 1.8 mM at the external and 9.4 mM at the internal side. By comparing the Km values at both sides of the membrane with the values found in intact mitochondria, a right-side-out orientation of the reconstituted phosphate carrier was concluded. Furthermore, the influence of various sulfhydryl reagents on the carrier was investigated. After modification with HgCl2, the phosphate carrier reveals a third (nonphysiological) unidirectional transport mode. This was characterized by a significantly reduced substrate specificity. In view of similar observations with several other mitochondrial carriers, these results again indicate that the phosphate carrier is a member of the postulated functional family of mitochondrial carrier proteins.