The transbilayer transport of lipids is essential in both eukaryotes and prokaryotes, yet the spontaneous rate of flipping is too slow to support cellular life. Physiologically relevant rates of flipping are achieved through the activity of two classes of lipid transporters. The first couples ATP hydrolysis to unidirectional lipid flipping in order to maintain asymmetric membranes, such as the eukaryotic plasma membrane. The second class of transporters facilitates bi-directional, ATP-independent movement of lipids across biogenic and specialized membranes. In a recent report (Menon et al. (2011) Curr. Biol. 21, 149-153) we identified opsin as the first ATP-independent flippase. We now report that opsin's flippase activity is constitutive, and not linked to its established function as a light sensor. We expressed thermostable variants of opsin in COS-7 cells and assayed activity of the purified proteins in a reconstituted system. We tested three structurally discrete signaling states of opsin: dark-adapted rhodopsin with the endogenous inverse agonist 9-cis retinal; the metarhodopsin II intermediate containing the agonist all-trans retinal in the constitutively active M257Y background; and the ligand-free light-adapted opsin. All constructs demonstrated rapid (t ½ < 10 s), ATP-independent flip-flop of zwitterionic phospholipid probes in both dark- and light-adapted conditions. These results suggest that the flippase activity of opsin, and likely other Type-A GPCRs such as the β1-adrenergic receptor which we also previously showed to have flippase activity, is localized to the relatively immobile transmembrane helices 1 - 4, or the amphipathic helix 8. These data, as well as the results of ongoing experiments on the flippase activity of dynamically constrained rhodopsin constructs, will be presented.Supported by NIH grant GM71041