To evaluate the role of antibiotic class in the rate of liberation of endotoxin during therapy for sepsis caused by gram-negative bacteria, we obtained serial blood samples from rabbits with sepsis caused by Escherichia coli and treated with chloramphenicol, gentamicin, or moxalactam. The concentrations of viable bacteria, free endotoxin, and total endotoxin in each blood sample were measured. In rabbits treated with chloramphenicol, the geometric mean levels of free endotoxin remained proportional to the geometric mean levels of bacteremia, a result indicating the absence of antibiotic-induced endotoxin liberation. In contrast, levels of free endotoxin increased rapidly while levels of bacteremia declined after treatment with gentamicin or moxalactam, a result indicating antibiotic-induced release of endotoxin. Despite similar rates of bacterial killing, mean levels of free endotoxin were as much as 20-fold higher in rabbits treated with moxalactam than in paired rabbits receiving gentamicin (P < .05). These results indicate that endotoxin liberation during therapy for sepsis caused by gram-negative bacteria is dependent upon the class of antibiotic administered and is not necessarily correlated with the rate of bacterial killing.