The carnitine carrier from rat liver mitochondria, solubilized in Triton X-100 and partially purified on hydroxyapatite, was identified and completely purified by specific elution from celite in the presence of cardiolipin. On SDS-gel electrophoresis, the purified celite fraction consisted of a single band with an apparent Mr of 32,500. When reconstituted into liposomes the carnitine transport protein catalyzed an N-ethylmaleimide-sensitive carnitine/carnitine exchange. It was purified 970-fold with a recovery of 43% and a protein yield of 0.04% with respect to the mitochondrial extract. The properties of the reconstituted carrier, i.e., requirement for a countersubstrate, substrate specificity and inhibitor sensitivity, were similar to those of the carnitine transport system as characterized in intact mitochondria.