The results of the present experiments have demonstrated that mitochondrial heme was transported to apocytochrome b5 incorporated into phospholipid vesicles in the presence of supernatant liver protein. The heme transfer depended on the concentration of supernatant protein, liposome-apocytochrome b5 and mitochondria, respectively. Omission of one of these components led to an almost complete loss of the transfer activity. The transport was a rapid reaction which showed an approximate linearity until 1.5 min at 37 degrees C after that became saturated. When the functional capacity was tested by the NADH-cytochrome c reductase system, the reconstituted cytochrome b5 expressed its complete original catalytic properties. The heme transfer activity was remarkably inhibited by KCN and NaN3, but was not affected by SH-reagent, such as N-ethylmaleimide and iodoacetate. ATP, EDTA and sodium fluoride had no effect. On the other hand, there was no evidence that supernatant protein participates in heme release from mitochondria or membrane fusion between mitochondria and phospholipid vesicles.