Because no simple in vitro test for cefotaxime (CTX) and desacetylcefotaxime (dCTX) synergy exists, and, because no disk susceptibility data is available for dCTX, we investigated the potential of antibiotic impregnated disks as a test for positive in vitro interaction. Results of this study indicate that all strains of Staphylococcus aureus tested demonstrated additive interactions using disks that contained 30 micrograms of dCTX. In contrast, it was necessary to use much smaller concentrations of each cephalosporin to demonstrate additive effects against Gram-negative bacteria. Zones of inhibition were greater for disks containing the combination compared to either CTX or dCTX alone, among all the Gram-negative genera tested; however, these differences were statistically significant only for Klebsiella pneumoniae and Escherichia coli (P less than 0.05). These results demonstrate that disk susceptibility testing is reliable for the CTX metabolite, dCTX, and suggests that this technique using disks impregnated with CTX and dCTX singly and in combination is a simple method with which to evaluate positive antimicrobial interactions in the clinical laboratory.