An image-based technique of fluorescence recovery after photobleaching (video-FRAP) was used to measure the lateral diffusion coefficients of a series of nine fluorescent probes in two model lipid bilayer systems, dimyristoylphosphatidylcholine (DMPC) and DMPC/cholesterol (40 mol%), as well as in human stratum corneum-extracted lipids. The probes were all lipophilic, varied in molecular weight from 223 to 854 Da, and were chosen to characterize the lateral diffusion of small compounds in these bilayer systems. A clear molecular weight dependence of the lateral diffusion coefficients in DMPC bilayers was observed. Values ranged from 6.72 x 10(-8) to 16.2 x 10(-8) cm2/s, with the smaller probes diffusing faster than the larger ones. Measurements in DMPC/cholesterol bilayers, which represent the most thorough characterization of small-solute diffusion in this system, exhibited a similar molecular weight dependence, although the diffusion coefficients were lower, ranging from 1.62 x 10(-8) to 5.60 x 10(-8) cm2/s. Lateral diffusion measurements in stratum corneum-extracted lipids, which represent a novel examination of diffusion in this unique lipid system, also exhibited a molecular weight dependence, with values ranging from 0.306 x 10(-8) to 2.34 x 10(-8) cm2/s. Literature data showed that these strong molecular weight dependencies extend to even smaller compounds than those examined in this study. A two-parameter empirical expression is presented that describes the lateral diffusion coefficient in terms of the solute's molecular weight and captures the size dependence over the range examined. This study illustrates the degree to which small-molecule lateral diffusion in stratum corneum-extracted lipids can be represented by diffusion in DMPC and DMPC/cholesterol bilayer systems, and may lead to a better understanding of small-solute transport across human stratum corneum.