To investigate the changes of gut microflora and endotoxin levels in rats with acute liver failure (ALF) induced by D-galactosamine (GalN). Flora and endotoxin levels in the jejunum, ileum and colon in normal rats (group A) and rats with GalN -induced ALF were determined at 24 h (group B) or 48 h (group C) after GalN injection, as well as the endotoxin level in portal venous blood (PVB) and right ventricle blood (RVB) were determined by chromogenic limulus amoebocyte assay. Intestinal (jejunum, ileum, colon) lactobacillus count was statistically reduced in group B compared with those in group A (3.4+/-0.3 vs 4.9+/-0.3, 6.1+/-0.4 vs 8.0+/-0.3, 8.1+/-0.2 vs 9.3+/-0.2, P<0.001, P<0.001 and P<0.001 respectively) and recovered partially in the group C compared with those in the group B, whereas the count of Enterobacteriaceae in the jejunum, ileum and colon in group B was increased markedly compared with those in the group A (5.1+/-0.3 vs 3.6+/-0.2, 6.9+/-0.5 vs 5.3+/-0.3, 8.7+/-0.2 vs 7.6+/-0.1, P<0.001, P<0.05 and P<0.05 respectively) and restored partially in the group C compared with those in the group B. The endotoxin level in ileum was increased in the group B compared with those in the group A (111.3+/-22.8 vs 51.5+/-8.9, P<0.05). In addition, the endotoxin level in PVB was obviously increased in group B compared with that in the group A (76.8+/-9.1 vs 40.6+/-7.3, P<0.01) and reduced to the baseline at 48 h (group C). Severely disturbed gut flora in rats with GalN-induced acute liver failure plays an important role in the elevation of endotoxin level in PVB.