The antibacterial efficacy of the combination of amoxicillin and cefotaxime was assessed against 50 clinical strains of Enterococcus faecalis. For 48 of 50 strains, the MIC of amoxicillin that inhibited 50% of isolates tested decreased from 0.5 microgram/ml (range, 0.25 to 1 microgram/ml) to 0.06 microgram/ml (range, 0.01 to 0.25 microgram/ml) in the presence of only 4 micrograms of cefotaxime per ml. Alternatively, the MIC of cefotaxime that inhibited 50% of isolates tested decreased from 256 micrograms/ml (range, 8 to 512 micrograms/ml) to 1 micrograms/ml (range, 0.5 to 16 micrograms/ml) in the presence of only 0.06 microgram of amoxicillin per ml. For JH2-2, a reference strain of E. faecalis, the MICs of amoxicillin, cefotaxime, and amoxicillin in the presence of cefotaxime (4 micrograms/ml) were 0.5, 512, and 0.06 microgram/ml, respectively. By using a penicillin-binding protein (PBP) competition assay, it was shown that with cefotaxime, 50% saturation of PBPs 2 and 3 was obtained at very low concentrations (< 1 microgram/ml), while 50% saturation of PBPs 1, 4, and 5 was obtained with > or = 128 micrograms/ml. With amoxicillin, 50% saturation of PBPs 4 and 5 was obtained at 0.12 and 0.5 microgram/ml, respectively. Therefore, the partial saturation of PBPs 4 and 5 by amoxicillin combined with the total saturation of PBPs 2 and 3 by cefotaxime could be responsible for the observed synergy between these two compounds.