Desacetyl-cefotaxime is the main cefotaxime metabolite. Its antibacterial activity is less than that of the parent molecule, but the combination of cefotaxime and desacetyl-cefotaxime is often synergistic. We analysed the hydrolysis of desacetyl-cefotaxime, in comparison with cefotaxime, by 10 beta-lactamases, mostly cephalosporinases, isolated from various Gram-negative species. From partially purified beta-lactamase preparations of high specific activity, we determined the maximum rates of hydrolysis (Vm) and the Michaelis constants (Ki and Km when possible). It appeared for 7 beta-lactamases the rate of desacetyl-cefotaxime hydrolysis was 2.1 times greater than that of cefotaxime, in terms of geometrical mean value, but with 3 enzymes no hydrolysis was shown for one compound. For 8 enzymes, the Ki, and sometimes Km, of desacetyl cefotaxime are 39 times higher than those of cefotaxime (geometric mean value), which corresponds to a lower affinity.