Two methods (cholate dialysis and cholate gel filtration) used to incorporate cytochromes P450 (P450s) and reductase into unilamellar phospholipid vesicles were compared with a standard reconstituted system (SRS) in which the proteins were reconstituted with preformed liposomes. Both cholate dialysis and gel filtration methods were comparable in their ability to physically incorporate reductase and either CYP2B4 or CYP1A2 into phospholipid, as determined by the elution of enzymes in the void volume using size exclusion chromatography (mol. wt. cutoff -5,000,000). Incorporation of these proteins was more efficient with both cholate methods than when reductase and P450 were mixed with preformed vesicles (SRS). Using either cholate method, more than 85% of the P450 was physically incorporated into the phospholipid vesicles, whereas less than 40% of the P450 was physically incorporated into the phospholipid vesicles using the SRS. Catalytic activities of the vesicular preparations of reductase and either CYP1A2 or CYP2B4 also were significantly higher than those resulting from the SRS using dilaurylphosphatidylcholine. Although both cholate dialysis and gel filtration methods improved protein incorporation when compared with preincubation of proteins with preformed liposomes, reductase incorporation was dependent on the relative amount of reductase used. Reductase incorporation was complete at a 0.2:1 reductase/P450 ratio; however, the efficiency of incorporation decreased to less than 50% at equimolar reductase/P450. Interestingly, reductase incorporation was higher in the presence of CYP1A2 than with CYP2B4. Both cholate methods resulted in the loss of a proportion of spectrally detectable carbon monoxyferrous P450, resulting from incubation of the proteins with detergent.