The aim of this investigation was to assess the applicability of lipid bilayer alteration using a combination of isopropyl myristate (IPM) and glyceryl monocaprylate (GEFA-C₈) to the enhancement of pentazocine (PTZ) permeation through hairless mouse skin. The skin permeability of PTZ was enhanced by increasing the concentration of GEFA-C₈ up to 10% w/w in combination with IPM. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and small angle X-ray diffraction (SAXD) were carried out to analyze the effects of these enhancers on the biophysical properties of the stratum corneum (SC) of the skin, and on the permeation of PTZ. ATR-FTIR studies revealed that IPM/GEFA-C₈ induced higher CH₂ stretching frequencies of SC lipids than IPM alone. SAXD showed the disappearance of long lamellar diffraction of SC lipids with IPM/GEFA-C₈, resulting in a complete loss of order of the SC lipid bilayers. When 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI), a hydrophobic fluorescence probe, was applied in IPM alone, the amount of DiI which penetrated into the intercellular space of the SC was very low, but this was markedly increased when DiI was applied in IPM/GEFA-C₈. These results indicate that the synergistic effects of IPM and GEFA-C₈ enhance transdermal permeation of PTZ by disrupting SC lipids.