Coproporphyrinogen oxidase (EC 1.3. 3.3), protoporphyrinogen oxidase (EC 1.3. 3.4), and ferrochelatase (EC 4.99. 1.1) catalyze the terminal three steps of the heme biosynthetic pathway. All three are either bound to or associated with the inner mitochondrial membrane in higher eukaryotic cells. A current model proposes that these three enzymes may participate in some form of multienzyme complex with attendant substrate channeling (Grandchamp, B., Phung, N., & Nordmann, Y., 1978, Biochern. J. 176, 97-102; Ferreira, GC, et al., 1988, J. Bid. Chem. 263, 3835-3839). In the present study we have examined this question in isolated mouse mitochondria using two experimental approaches: one that samples substrate and product levels during a timed incubation, and a second that follows dilution of radiolabeled substrate by pathway intermediates. When isolated mouse mitochondria are incubated with coproporphyrinogen alone there is an accumulation of free protoporphyrin. When Zn is added as a substrate for the terminal enzyme, ferrochelatase, along with coproporphyrinogen, there is formation of Zn protoporphyrin with little accumulation of free protoporphyrin. When EDTA is added to this incubation mixture with Zn, Zn protoporphyrin formation is eliminated and protoporphyrin is formed. We have examined the fate of radiolabeled substrates in vitro to determine if exogenously supplied pathway intermediates can compete with the endogenously produced compounds. The data demonstrate that while coproporphyrinogen is efficiently converted to heme in vitro when the pathway is operating below maximal capacity, exogenous protoporphyrinogen can compete with endogenously formed protoporphyrinogen in heme production. Likewise, exogenously supplied protoporphyrin efficiently competes with protoporphyrinogen in the formation of heme. These two lines of experiments strongly suggest that an obligatory channeling complex does not exist for the terminal three enzymes of the heme biosynthetic pathway, but that the enzymes function in a manner such that significant accumulation of intermediates does not normally occur.