Pharmacokinetics was investigated when ampicillin (ABPC) (50 mg/kg) and dibekacin (DKB) (5 mg/kg), in combination with pre- or post-treatment, were intravenously injected to the rat or rabbit. 2. The antibiotics in the samples were separated by the paper-electrophoretic technique and then concentrations were determined by the cup thin-layer plate method using Bacillus subtilis ATCC 6633 as the test organism. 3. The biological half-life of DKB was prolonged in pretreatment with ABPC and that of ABPC was shortened in pretreatment with DKB. An initial level of ABPC was elevated. Similar tendency was observed in both of the rat and rabbit. 4. Urinary excretion rates of both antibiotics in the combining group tended to decrease compared with the single administration group. 5. Binding of ABPC to serum proteins was competitively inhibited by DKB. Binding of DKB to serum proteins increased.