The contribution of aerobic and anaerobic gram-negative intestinal bacteria to the release of endotoxin in the intestinal tract was investigated during oral administration of various nonabsorbable antimicrobial drugs to C3H/Law mice. The intestinal endotoxin release was studied by determination of the endotoxin concentration in faecal supernatants with the Limulus amebocyte lysate assay. Selective elimination of aerobic gram-negative bacteria by oral treatment with polymyxin, aztreonam or temocillin resulted in a reduction of the endotoxin concentration of faecal supernatants to 10% of the untreated control. Further decrease of the endotoxin concentration to 1% was achieved by total decontamination of the intestinal tract by oral cephalothin/neomycin treatment. Endotoxin determination with the Limulus amebocyte lysate assay appeared to be unaffected by the antibiotics present in the faecal supernatants after oral treatment. On basis of these experiments, it is concluded that in mice 90% of the faeces derived endotoxin can be ascribed to release of endotoxin by intestinal aerobic gram-negative bacteria.