Laser scanning confocal microscopy (LSCM) has been used to visualize and quantitate the penetration of a model, anionic, fluorescent compound (calcein) along the iontophoretic transport pathways within hairless mouse skin. The LSCM technique permits optical sectioning of full-thickness, unfixed tissue, thereby avoiding poor image quality due to blurring from out-of-focus fluorescence, and obviating artifactual redistribution of the permeant during processing. Simple measurements of the approximate flux of the probe across hairless mouse skin were also made using standard in vitro diffusion cell methodology and a fluorometric assay. Most importantly, LSCM imaging strategies were developed to overcome depth-dependent sensitivity problems. These visualization studies showed that iontophoresis of calcein into hairless mouse skin enhanced delivery, particularly via follicular structures, to significant depths into the barrier. Nonfollicular transport was also apparent, especially at more superficial levels. Quantitative analyses of the LSCM images showed that, although significant nonfollicular transport occurs, the efficiency of the follicular pathway, when the relative surface area is taken into account, is considerable. Overall, therefore, this work contributes significantly to the ultimate goal of fully comprehending the mechanism(s) of iontophoretic drug delivery across the skin.