CO-heme interactions with sperm whale apomyoglobin (apoMb) and model membrane systems were examined by spectroscopic and kinetic techniques. The binding of heme to apoMb appears to be a simple bimolecular process at relatively low apoprotein concentrations. The association rate constant, 3 × 10\sp7 M\sp−1s\sp−1, was measured directly at 10\sp∘C. The circular dichroism spectra for newly reconstituted Mb was initially decreased when compared to native Mb. The half time for equilibration to the native state can range from several hours to days depending on the pH, ligand, and oxidation state. The rate constants for the association or dissociation of oxygen or carbon monoxide to newly formed Mb were identical to native Mb. Kinetic heterogeneity was observed in long-chain isonitrile binding. These results suggest that although rapid uptake of heme by the apoprotein can result in two orientations, there are no apparent changes in the physiological properties of myoglobin. The effects of vesicle composition on heme binding to membranes were probed using thirty separate lipid mixtures. A sharp decrease in the rate of heme binding to liposomes was observed as the lipid vesicles changed form liquid-crystalline to gel phase. The addition of dicetyl phosphate, which has a negative charge at neutral pH, generally decreased the overall rate and affinity of the vesicles for heme binding. The rate and extent of heme uptake in unsaturated lecithins is unaffected by cholesterol content at levels up to 40% per mole. In contrast, the affinity of saturated dimyristoylphosphatidylcholine (DMPC) vesicles for heme dramatically decreased with increasing cholesterol content. This effect appears to be related to the influence of cholesterol on the DMPC phase transition temperature (T\sbm). Although there is some variation, most lecithins with low T\sbm values have an overall equilibrium partition constant $\approx$5 × 10\sp5 and association and dissociation rate constants $\approx$3 × 10\sp6 s\sp−1 and 7 s\sp−1, respectively, at 30\sp∘C. Association, dissociation, column chromatography, and temperature dependence studies of CO-heme binding to liposomes indicate that the rate of heme transmembrane movement is slow and dependent on phosphatidylcholine acyl-chain length. The rate increases sharply at the phase transition temperature.