In a previous study from our laboratory (Drug Metab Dispos 34: 660-666, 2006), we found several limitations with published methods (cholate gel filtration and cholate dialysis) for the incorporation of cytochromes P450 and P450 reductase into phospholipid vesicles. We found that a significant proportion of reductase was not incorporated in the vesicles when the amount of reductase was equal to or greater than that of CYP2B4 in the systems reconstituted with phosphatidylcholine. Furthermore, implementation of these methods compromised the ability of the CYP2B4 to form a ferrous carbon monoxy complex. In the current study, a comparison of results using the detergent-dialysis method with five similar detergents having the "bile salt" ring structure showed that glycocholate results in the greatest incorporation of reductase and the least loss in the ferrous carbon monoxy CYP2B4 complex. The method is further improved by using Bio-Beads SM-2 to remove detergent instead of the lengthy dialysis procedure or size exclusion chromatography that significantly dilutes the protein and lipid concentrations of the preparation. The method is shown to be applicable over a range of lipid/CYP2B4 ratios, and by using assay methods for total lipid, reductase, and CYP2B4, this improved reconstitution method resulted in increased incorporation efficiencies while minimizing the protein degradation inherent with these procedures.