When rhodopsin is incorporated into the saturated short-chain phospholipid dimyristoylphosphatidylcholine, photolysis of the protein results in an abnormal sequence of spectral transitions, and the dominant product of metarhodopsin I decay is free retinal plus opsin [Baldwin, P. A., & Hubbell, W. L. (1985) Biochemistry (preceding paper in this issue)]. By incorporation of rhodopsin into a series of phosphatidylcholines of defined composition, we have determined the properties of the lipid environment that are responsible for the altered spectral behavior. Metarhodopsin II is not found in appreciable amounts in bilayers containing acyl chains that are too short (14 or fewer carbon atoms in length), in the presence of only n-alkyl chains, or below the characteristic phase-transition temperature of recombinant membranes. Double bonds are not required for the formation of the metarhodopsin II intermediate, as it is observed in diphytanoylphosphatidylcholine recombinants.