In rat serum as well as in human serum and bile after injection of cefotaxime (CTX), the parent compound and the active metabolite desacetyl cefotaxime (dCTX) have been demonstrated by quantitative analysis with high-performance liquid chromatography (HPLC). Simultaneous determination of CTX by bioassay using test organisms sensitive to both CTX and dCTX resulted in spuriously high concentration readings of CTX. Using dCTX insensitive test organisms concentrations of cefotaxime obtained by agar diffusion test and by assay with HPLC were highly correlated. In human serum and bile after i.v. injection of 2 g CTX, as may be administered therapeutically, high concentrations of dCTX were observed. dCTX has a longer elimination half-life than the parent molecule. It is concluded that the measurement of CTX in biological fluids should be performed by HPLC or by bioassay with a selective test organism in order to obtain correct pharmacokinetic parameters.