Infrared (IR) microscopic imaging is used, in a series of proof-of-principle experiments to map the spatial distribution of two penetration enhancers, dimethylsulphoxide (DMSO) and propylene glycol, in skin. The current instrumentation utilizes a 64×64 array of IR detectors imaged at the focal plane of an IR microscope, each collecting a complete mid-infrared spectrum of the skin section on each pass of the interferometer. The spatial area sampled by each element in the array is ~6.3×6.3 μm. Any spectral parameter (e.g., arising from lipid or protein vibrational modes of the endogenous tissue or the exogenous component) may be quantitatively analyzed across the entire array of 4096 spectra, thereby generating an IR spectroscopic image of that particular parameter throughout the sample. The images directly reveal the spatial heterogeneity of the protein and lipid distributions. In transverse slices of skin, the depth dependencies of the spatial distribution of triglyceride and protein have been monitored, and compared to those of the exogenous penetration enhancers. Images of both DMSO and propylene glycol suggest that each penetrates the skin to a depth of at least 1 mm (under our experimental protocols), and reveals a spatial distribution that is essentially coincident with the protein constituents of the skin. These results demonstrate that IR microscopic imaging has great potential for mechanistic studies of topical, dermal, and transdermal delivery.