Detailed models of solute transport through the stratum corneum (SC) require an interpretation of apparent bulk diffusion coefficients in terms of microscopic transport properties. Modern microscopy techniques provide a tool for evaluating one key property—lipid pathway tortuosity—in more detail than previously possible. Microscopic lipid pathway measurements on alkali expanded human SC stained with the lipid-soluble dyes methylene blue, Nile red, and oil red O are described. Brightfield, differential interference contrast, fluorescence, and laser scanning confocal optics were employed to obtain 2-dimensional (2-D) and 3-dimensional (3-D) images. The 2-D techniques clearly outlined the corneocytes. Confocal microscopy using Nile red yielded a well-delineated 3-D structure of expanded SC. Quantitative assessment of the 2-D images from a small number of expanded SC samples led to an average value of 3.7 for the ratio of the shortest lipid-continuous pathway to the width of the membrane. This was corrected for the effect of alkaline expansion to arrive at an average value of 12.7 for the same ratio prior to swelling.