The interaction of cephalosporin antibiotics with renal cortex of rats has been examined in vitro by means of cortical slice uptake and binding assay to plasma membranes and other subcellular fractions, including the comparative study with p-aminohippurate. The uptakes of cephalexin and cephaloridine by cortical slices were concentrative, and were strongly inhibited in the presence of 2,4-dinitrophenol, ouabain, nitrogen gas and probenecid. In the case of cefazolin, the degree of concentrative uptake and influence of inhibitors were low. Cephalexin, cephaloridine and p-aminohippurate specifically bound to the basolateral membranes, compared to brush border membranes and other subcellular fractions. Cefazolin binding to basolateral membranes was relatively small. These results suggest that the specificity of cephalexin and cephaloridine bindings to the basolateral membranes could be related to the interaction with the organic acid transport system at the antiluminal side. Thus, an examination of cephalosporin interaction with plasma membranes from renal cortex could offer an appropriate in vitro model system to study the renal transport of these antibiotics.