The in vitro activities of cefotaxime (CTX), desacetylcefotaxime (DES), cefuroxime (CXM) and ceftizoxime (CTZ) were compared against 272 recent clinical isolates and the effect of CTX and DES in combination was studied using agar dilution checkerboards. CTX and CTZ had similar activity, inhibiting 90% of Escherichia coli, Proteus spp., Klebsiella spp. and Providence spp. at 0.0075 mg/l. Ninety percent of Enterobacter spp. and Serratia spp. were inhibited by 1 mg/l of CTX and CTZ. Ninety percent of staphylococci were inhibited by 4 mg/l of CTX, although CTZ was significantly less active against strains of methicillin-resistant Staphylococcus epidermidis. Pseudomonas aeruginosa and enterococci had MICs for CTX of 32 and 128 mg/l respectively. DES had similar activity to CXM with MICs 2 to 5 dilutions less than CTX and CTZ. In checkerboard tests, synergy (FIC less than 0.5) between CTX and DES was demonstrated against 34% of isolates overall, with antagonism (FIC greater than 1) in 24%. CTX and DES were synergistic against 72% of staphylococci and 50% of enterococci. They were antagonistic against 81% of Enterobacter spp. and 62% of Serratia spp. although both species were susceptible to CTX alone. There was an additive effect or slight antagonism with 90% of Ps. aeruginosa. Other investigators have reported lower incidences of antagonism between CTX and DES: this could be due to variations among the collections of clinical isolates or methodologies used. The relevance of the in vitro interaction between CTX and its metabolite to the antimicrobial activity of CTX in vivo remains to be demonstrated.