A variety of techniques are currently in use for preparing protein-containing lipid vesicles known as proteoliposomes. However, the functionality of membrane protein in proteoliposomes prepared by various techniques has rarely been evaluated directly. We prepared rhodopsin-containing proteoliposomes consisting of asolectin or native retinal rod outer segment disk lipids using n-octyl beta-d-glucopyranoside and the detergent dialysis (DD) and rapid dilution (RD) techniques and measured the activity of rhodopsin using equilibrium UV/vis and flash photolysis spectroscopy. A significant difference in rhodopsin activity was observed in proteoliposomes prepared by these techniques. The equilibrium constant of metarhodopsin I-metarhodopsin II is 30-45% higher, and the apparent rate constant of MII formation is up to 3-fold faster in proteoliposomes prepared by RD vs DD. The DD technique produced larger yet more heterogeneous vesicles, while the RD technique yielded smaller and more homogeneous vesicles, as determined by electron microscopy and isopicnic centrifugation. Both proteoliposomes and empty lipid vesicles lacking rhodopsin were formed in the DD preparation, while only proteoliposomes were formed in the RD preparation. Under identical conditions, proteoliposomes prepared by RD have a higher L/P ratio, which is consistent with the higher level of rhodopsin activity in RD proteoliposomes. Overall, the results presented here suggest that the RD technique has an advantage over the DD technique in terms of preserving optimal rhodopsin activity and controlling the lipid to protein ratio in the final proteoliposomes.