The permeability of fosfomycin (FOM), which is a low molecular weight and water-soluble antibiotic, across rat and rabbit jejunal membrane was investigated on a paracellular route and a transcellular route separated by voltage-clamp method. It was determined that FOM permeates through these two routes in both animals. Under a physiologically normal condition, the transmucosal electrical potential difference (about 2 mV), the contribution of the transcellular route to FOM membrane permeability was greater than that of the paracellular route. The transcellular permeability was significantly reduced in the presence of inorganic phosphate. The inhibitory effects of the inorganic phosphate coincided with our reports on FOM uptake by rat jejunal brush-border membrane vesicles in vitro and on FOM absorption by rat jejunal single-pass perfusion in situ.