Properties of single fluorescence-labeled lipids in a fluid phospholipid membrane were analyzed using sensitive fluorescence microscopy. The high signal-to-noise ratio for detection of individual fluorescence-labeled molecules of 5-70 permitted detailed study of their photophysical parameters. From single fluorophores a signal of 49 counts/ms was detected when excited above their saturation intensity of 7.6 kW/cm(2). These data, obtained at the level of individual fluorophores, are found to agree quantitatively with predictions from a conventional three-level model using known ensemble-averaged rate constants. Light-induced photochemistry of single fluorophores was found to occur as a sudden disappearance of their fluorescence characterized by a photobleaching efficiency of 9 x 10(-6). These photophysical properties of the fluorescence label permitted analysis of the motion of individual lipid molecules. Lipid motion was tracked at a rate of up to 66 images per second. The high fluorescence image-contrast allowed for determination of lipid positions with a precision of 30 nm. For short length-scales the observed trajectories were described by a random two-dimensional motion with a lateral diffusion constant of 1.42 x 10(-8) cm(2)/s. Free diffusion was found to be restricted to length-scales of 100 nm, attributed to membrane defects.