The purpose of the present study was to characterize the passive permeation of tetracyclines in the parallel artificial membrane permeation assay (PAMPA). Tetracyclines exist as zwitterion at physiological pH. The PAMPA membrane was prepared by impregnating a phospholipid/decane solution to a filter support. The permeation coefficient (Pe) of tetracycline (TC) was markedly affected by the lipid composition of the PAMPA membrane. No permeation was observed when phospholipid was not added (pure decane membrane, Pe < 0.05 × 10-6 cm/sec). With the addition of 2 % PC, little or no increase in Pe was observed. The addition of 1 % PE increased the Pe value more than tenfold. The addition of 2 % soybean lecithin containing phosphatidylinositol (PI) and phosphatidic acid (PA) increased the Pe value to above 4 × 10-6 cm/sec. The Pe value was further increased to 15 × 10-6 cm/sec by increasing the concentration of soybean lecithin from 2 to 10 %. The Pe value showed pH and temperature dependence, whereas it was not affected by the ionic strength, TC concentration, and ion-pair transport inhibitors. A weak correlation was observed between the Pe values and octanol-buffer distribution coefficients of tetracyclines. These results suggest that inter-molecular interactions between TC and PE, PI and/or PA facilitate the passive diffusion of TC across the PAMPA membrane.