Revised Manuscript Received July 8, 1993* abstract: How lipophilic acids move across membranes, either model or biological, is the subject of controversy. We describe experiments which better define the mechanism and rates in protein-free phospholipid bilayers. The transbilayer movement of lipophilic acids [fatty acids (FA), covalently-labeled FA, bile acids, and retinoic acid] was monitored by entrapping pyranin, a water-soluble, pH-sensitive fluorescent molecule to measure pH inside unilamellar vesicles [Kamp, F., & Hamilton, J. A.(1992) Proc. Natl. Acad. Sci. USA 89, 11367-11370]. Equations for the pseudo-unimolecular rate constants for transbilayer movement of un-ionized (/cFAh) and ionized (&fa-) acids are derived. All FA studied (octanoic, lauric, myristic, palmitic, stearic, oleic, elaidic, linoleic, linolelaidic, and arachidonic) and retinoic acid exhibited rapid transbilayer movement (ti/2< 1 s) via the un-ionized form across small unilamellar egg phosphatidylcholine (PC) vesicles. FA produced by phospholipase A2 in the outer leaflet of PC vesicles equilibrated rapidly to the inner leaflet. Ionized FA showed enhanced transbilayer movement (£ Fa--0.029 s_1) in the presence of equimolar valinomycin. The three FA analogues [12-(9-anthroyloxy) stearic acid, 5-doxylstearic acid, and 1-pyrenenonanoic acid] moved across PC bilayers via the un-ionized form; except for the anthroyloxy FA (£ Fah= 4.8 X 10~ 3 s_1), the rates were too fast to measure(fi/2< 1 s). The rate for cholic acid (CA) transbilayer movement was slow (&cah=