To prove the free-ligand hypothesis for extravascular equilibration and tissue binding mechanism of β-lactam antibiotics, the microdialysis technique has been employed for the lung, the muscle and the liver in rats. Cefminox, a cephem antibiotic, and SY5555, a new penem antibiotic, were used in the present study. During the constant infusion of each antibiotic with simultaneous infusion of antipyrine, the microdialysis studies were performed and the dialysate concentrations were determined. The dialysate concentration was extrapolated to the in vivo unbound concentration in tissue interstitial fluids (C_{isf, u}) according to the extrapolation method which was derived from the clearance concept. This extrapolation method incorporates the effective dialysis coefficient of a reference compound, antipyrine, which is used to correct the difference between in vivo and in vitro permeabilities of microdialysis fiber. The values of C_{isf, u} values for cefminox and SY5555 in the lung, muscle and liver were close to the unbound concentrations in the venous plasma leaving these organs. Furthermore, good coincidences were obtained between the unbound concentrations of SY5555 in lung and muscle interstitial fluids estimated from the total concentrations in homogenized tissues and those extrapolated by the microdialysis studies. Consequently, the present microdialysis studies provided the in vivo evidence that 1) the free-ligand hypothesis for extravascular equilibration of β-lactam antibiotics is true, and that 2) β-lactam antibiotics are restricted in the interstitial space in a noneliminating organ and bind only with albumin existing in this space.