Ro 23-9424 is a broad-spectrum antibacterial agent consisting of a cephalosporin (desacetylcefotaxime) linked through an ester bond to a fluoroquinolone (fleroxacin). Its activity against mutants of Escherichia coli TE18 resistant to both antibacterial components was examined. E. coli TE18 overproduces the AmpC beta-lactamase and is resistant to several cephalosporins, including desacetylcefotaxime (MIC, 50 micrograms/ml), although it is still susceptible to Ro 23-9424 (MIC, 0.2 microgram/ml). Thirty-five spontaneous, two-step mutants of E. coli TE18 which were resistant to fleroxacin (MIC, 50 micrograms/ml) were isolated. In the mutants, replicative DNA biosynthesis (permeabilized cells) was resistant to fleroxacin, and some mutants had porin abnormalities. However, all remained susceptible to Ro 23-9424 (MIC, 0.5 microgram/ml). Examination of beta-lactamase activity in the parent strain revealed that it hydrolyzes desacetylcefotaxime more rapidly than it does Ro 23-9424. Thus, Ro 23-9424 may be less susceptible to the gram-negative, chromosomal beta-lactamases that hydrolyze several broad-spectrum cephalosporins and may be effective in cases in which neither of its two components is active.