A highly efficient in vitro system was established for measuring by high performance liquid chromatography the formation of holocytochrome c by yeast mitochondria. Holocytochrome c formation required reducing agents, of which dithiothreitol was the most effective. With biosynthetically made, pure Drosophila melanogaster apocytochrome c and Saccharomyces cerevisiae mitochondria, the activity of cytochrome c heme lyase amounted to about 800 fmol min-1 mg-1 mitochondrial protein. The kinetics were typical Michaelis-Menten (Km approximately 1 nM), as were those of mitoplasts with broken outer membranes (Km approximately 3 nM). As tested with mitoplasts, holocytochromes c from a range of species were found to be competitive inhibitors of heme lyase at physiological concentrations, providing a mechanism for controlling this concentration in vivo. Apocytochrome c associated with yeast mitochondria in two phases of Kd approximately 2 x 10(-10) and 10(-8) M, respectively, whereas mitoplasts had lost the high affinity binding. A site-directed mutant of apocytochrome c (lysines 5, 7, and 8 replaced by glutamine, glutamic acid, and asparagine) was found to be converted to holocytochrome c (Km approximately 3.3 nM; maximal activity unchanged), even though the mutations completely eliminated the high affinity binding. Thus, the high affinity binding of apocytochrome c to mitochondria is not directly related to holocytochrome c formation.