An assay was developed to study the spontaneous transfer and transbilayer movement (flip-flop) of lipid analogs labeled with the fluorescent fatty acid, 5-(5,7-dimethyl BODIPY)-1-pentanoic acid (C5-DMB-) in large unilamellar lipid vesicles comprised of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC). The assay is based on the concentration-dependent changes in fluorescence intensity that occur when donor vesicles containing a C5-DMB-lipid are mixed with nonfluorescent acceptor vesicles. A kinetic model was developed to describe the time-dependent changes in concentration of a lipid undergoing both spontaneous transfer between unilamellar vesicles and transbilayer movement within the vesicle membranes, and a mathematical solution was obtained. Data were obtained using C5-DMB-labeled analogs of sphingomyelin (C5-DMB-SM), ceramide (C5-DMB-Cer), phosphatidylcholine (C5-DMB-PC), and diacylglycerol (C5-DMB-DAG), and kinetic parameters for each lipid were determined using a nonlinear least-squares fitting program. The half-times for interbilayer transfer of the lipids were C5-DMB-SM (21 s) < C5-DMB-PC (350 s) approximately C5-DMB-Cer (400 s) << C5-DMB-DAG (100 h). C5-DMB-Cer (t1/2 approximately 22 min) and C5-DMB-DAG (t1/2 approximately 70 ms) exhibited rapid spontaneous transbilayer movement, while C5-DMB-SM (t1/2 approximately 3.3 h) and C5-DMB-PC (t1/2 approximately 7.5 h) moved across the bilayer very slowly. These results provide a basis for interpreting the behavior of these lipid analogs in cells.