We evaluated the in vitro interaction of clinically attainable concentrations of cefotaxime and ceftriaxone with chloramphenicol against 26 clinical isolates of gram-negative rods, group B streptococci, and Staphylococcus aureus. Cefotaxime and ceftriaxone were bactericidal against all 26 organisms (MBC, 0.03 to 4 μg/ml). Chloramphenicol was bacteriostatic against 24 organisms (MBC, ≥ 32 μg/ml) and bactericidal against two Escherichia coli isolates (MBC, 8 μg/ml). Checkerboard testing showed chloramphenicol to be antagonistic to the bactericidal activity of cefotaxime and ceftriaxone for all 24 bacteria for which chloramphenicol was bacteriostatic. Time kill curves for selected strains of E. coli and group B streptococci for which chloramphenicol was bacteriostatic showed antagonism of chloramphenicol to both cephalosporins. The combination of chloramphenicol with either cephalosporin was antagonistic in cases in which chloramphenicol was bacteriostatic against the above organisms and should be avoided in the treatment of infections caused by such organisms if bactericidal therapy is desired.