We developed a rapid and precise high performance liquid chromatographic method (HPLC) for the determination of azlocillin and its metabolites penicilloate and penilloate in serum and tissue as well as in vivo as in vitro. The linear relationship (r greater than 0.99) of determination ranged between 0.05 and 10 micrograms. No interference with other serum components was observed. The metabolism of azlocillin in vitro was analysed in serum. Within 24 h the amount of penicilloate increased from 1.5% up to 18% and from penilloate up to 1% respectively. Buffer controls revealed a significant lower metabolism (4.7% penicilloate, 0.9% penilloate). These data suggest that the degradation of azlocillin does not only depend on bacterial beta-lactamase activity but is influenced by enzymatic activities within serum and human tissue.