The site of action of surfactants incorporated in ointment bases was investigated with the aid of radioactive tracer techniques. Representative samples of the two major types of nonionic surfactants, polysorbate 85 and polyoxyethylene (10) oleyl ether were incorporated into white petrolatum and applied daily to rabbit skin clipped free of hair. After 4 days of treatment the excised epidermis was analyzed for phospholipids, nucleic acids, and the in uitro incorporation of 32P into phospholipids and the trichloroacetic acid (TCA)-soluble fractions. Results indicated that the surfactant treatment induced an increase in the epidermal phospholipid content, and a decrease in the phosphorus content of the TCAsoluble fraction. The incorporation of 32P into phospholipids and the TCA-soluble fraction was approximately 2-2.5 times greater in the surfactant-treated skin samples than in control samples. These and other available data suggest that the site of action of surface active agents is in the biological membranes. These surfactants may interact with membranes by either rupturing them, replacing certain phospholipids present in the lipid micelles, or inducing configurational changes in these micelles.