The aim of this study was to determine the incidence and levels of the gram negative bacterial endotoxin, and to investigate the effects of antiendotoxemic treatment along with operation. Thirty bitches with pyometra were used in this study. The bitches were randomly allocated into two groups as Group I (treated with polymyxin E-ampisillin) and Group II (treated with ampisillin). Samples for bacteriological examination were taken from vagina, vesica urinaria and uterus of all bitches. Blood samples were collected to evaluate biochemical, haematological tests and serum toxine levels before the operation and on 7 days after operation. Gelation was carried out to determine the gram negative bacterial endotoxin level with E-toxate test kit in accordance with gelation system. Gram negative bacteria were isolated 66% in Group I and 80% Group II. Escherichia coli were dominantly (63.4%) isolated from the uteri of 30 bitches with pyometra. Before the operation, serum toxin levels were found in Group I (n=10) 1.2 +/- 0.4 EU/ml and in Group II (n=12) 0.9 +/- 0.2 EU/ml. Five bitches in Group I and 3 bitches in Group II died on the third day after operation. Survival rate was a positive correlation with toxin levels (r=0.727; P<0.01) and BUN (r=0.539; P<0.01), creatinin (r=0.504, P<0.05) before operation in recovered and dead bitches. Serum toxin levels were not different between Group I (n=6; 0.14 +/- 0.02 EU/ml) and Group II (n=11; 0.19 +/- 0.01 EU/ml) 7 days after operation. Difference of toxin levels in both groups were different (P<0.05) between the preoperation period and the 7th day after operation. Before the operation and on the 7th day after the operation, in Group I difference between band neutrophil count and in Group II ALT, ALP, BUN, creatinin, RBC, WBC, Hct, band neutrophil and lenphocyte value were found significant (P<0.05). As a result, there was a positive correlation between toxin levels and BUN-creatinin values. Also, the cases that have the increased BUN and creatinin levels along with high toxin levels were trended towards to low survival rate. After operation, in cases that performed with ampicillin were acquired more effective results contrary to the hipothesis according to polymyxin E+ampicillin combination in bitches with pyometra.