Proteoliposomes loaded with varying levels of internal substrates were used in bisubstrate initial velocity studies to gain insight into the transport mechanism of the reconstituted chloroplast phosphate translocator. The kinetic response to trans substrates clearly indicated that the one-to-one exchange mediated by this translocator proceeds via a ping-pong type, and excluded a sequential type of reaction mechanism. It is also shown that reconstitution of the protein leads to an unidirectional orientation of the protein within the liposomes being orientated right-side-out with respect to chloroplasts. Different transport affinities were observed on either side of the membrane and only the outward-facing transport site of the translocator is able to bind inhibitors i.e. pyridoxal 5'-phosphate (PLP) and 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS).