The final steps in heme synthesis take place within mitochondria while the acceptor apoproteins are synthesized on the endoplasmic reticulum. When 14C-δ-aminolevulinic acid is used as a heme precursor in intact rats, measurable 14C-heme is found to be associated with the microsomes within 10 min of intraperitoneal injection. This rapid transfer of heme from mitochondria was studied in vitro using isolated rat liver mitochondria, and protoporphyrin IX and 59Fe as heme precursors. These mitochondria synthesize heme when suspended in whole cell sap and this is only partially reduced by substituting Sephadex G-25 filtered cell sap or sucrose. Mitochondria incubated in G-25 filtered cell sap or sucrose synthesize equivalent amounts of heme but those in sucrose export little heme into the surrounding medium. Heme export from mitochondria is dependent on protein in the suspending medium. In cell sap, heme is associated with multiple proteins and no single carrier was identified. Heme probably makes its way from mitochondria to microsomes via various protein carriers by nonspecific adherence. Microsomal apoproteins or other heme binding proteins then remove the heme from the intermediate carrier as the terminal step.