4 JOURNAL OF COSMETIC SCIENCE observed with Jeol 100SX operated at 80kV. The main steps of the freeze fracture electron microscopy technique are summarized in Figure 1. The median sizes of the oily droplets were determined from microscopic negatives. To localize benzophenone-4 in the emulsions, benzophenone-4 was assayed in the whole emulsions and in the aqueous phases obtained after ultracentrifugation. Benzophenone-4 was quantified using reversed-phase HPLC with a column of Novopack C18 at 285 nm. The mobile phase consisted of water with 1% perchloric acid and methanol (6:4). THE CUTANEOUS MEMBRANE Benzophenone-4 flux was determinated across ventral skin of male 6-7-week-old hairless rats (Iffa Credo). The skin was always freshly obtained and used full-thickness, with the subcutaneous fat carefully removed. IN VITRO PERMEATION STUDIES The in vitro drug release experiments were performed with static vertical diffusion cells with an exposure area of 2.54 cm 2. Five tenths of a gram of each emulsion was applied to the rat skin surface. The receiving fluid (9 ml) was phosphate buffer, pH = 7.4, with 5 % (w/w) polysorbate 80 to ensure sink conditions and with 0.5% (w/w) of antibiotics (streptomycin 10000 UI/ml and penicillin 10000 I•g/ml) to prevent microbiological proliferation. During permeation experiments, the receiver phase was constantly stirred by means of a magnetic bar. The receiver medium was collected over a period of 48 hours. For each system a mean of six cells was calculated. RESULTS AND DISCUSSION DETERMINATION OF THE EMULSION STRUCTURE The freeze-fracture electron microscopy consisted of observing and photographing the metallic replica of the fractured surface of the cryofixed sample. Shadows were cast by any topographical features of the specimen surface, and a three-dimensional impression of the surface was thus created. For emulsions with polysorbate 60, poloxamer 407, and acrylates/C•o_3o alkyl acrylate crosspolymer, all the fractured oily droplets and continuous aqueous phases were ho- mogeneous without relief. These three emulsions did not contain any liquid crystal structure (Figure 2). Micrographs of emulsions with triethanolamine stearate, sorbitan stearate and sucrose cocoate, and steareth-2/-21 clearly showed lameliar liquid crystals in the aqueous phase and vesicles with relief, which were oily droplets surrounded by one or more lameliar bilayers (Figure 3). Table II summarizes the emulsion structure, the medium size of oily droplets, and the distribution of benzophenone-4. The relatively amphiphilic character of benzophenone-4 may predispose it to associate with surfactant structures.