Electron paramagnetic resonance (EPR) imaging with the modulated field gradient technique is a novel method to investigate skin biophysical and biochemical properties employing specific nitroxide spin probes. Using this method, a distinct increase in polarity from epidermis towards lower dermis is observed with the spin label dit-butylnitroxide (DTBN). With proxylmaleimide a considerable increase in mobility is found, when epidermis is compared with dermal compartments. The effect of the natural antioxidant dihydrolipoate on skin membrane polarity was studied. Skin penetration of spin labeled dihydrolipoate was investigated by EPR imaging. The results indicate that dihydrolipoate also increases membrane polarity. The biophysical and biochemical changes in the epidermis and dermis as revealed by spatial imaging, provide indirect evidence for skin penetration of dihydrolipoate. This conclusion was supported by the finding that spin labeled derivatives of dihydrolipoate and lipoate were detected inside epidermis and dermis by EPR imaging. This study demonstrates the feasibility of EPR imaging to investigate pharmacodynamic and pharmacokinetic properties of spin labeled drugs in skin.