Tissue distribution of ampicillin following a single intravenous injection was studied in normal and functionally nephrectomized rabbits. Serum concentrations of ampicillin were compared with those obtained in subcutaneous tissue cage fluid, muscle (measured by a direct agar diffusion method) and muscle tissue fluid (obtained from implanted cotton threads). Considerable differences were found. Penetration of ampicillin into rabbit muscle was rapid and the elimination half-life was similar to that of serum. Concentrations in tissue cage fluid were low as compared with muscle tissue fluid levels, and penetration and elimination was slower. Results from experiments in functionally nephrectomized rabbits showed that a steady state between serum and muscle seemed to be attained after 15-30 min. These results were used to estimate the degree of protein binding of ampicillin in muscle tissue fluid.