Sepsis is the major cause of mortality in ICU. Microdialysis was introduced as a method to determine the content of the interstitial space. Metronidazole is a recognised antimicrobial agent which has high efficiency and a low rate of toxicity and resistance. The aims of the study were to evaluate the concentration of metronidazole in the plasma and in the muscle tissue, using microdialysis, in patients with septic shock and to compare it with the respective concentrations in healthy volunteers and with the MIC90 values for the B. fragilis group. The group of septic shock group consisted of six male patients admitted to the general ICU with diagnosed septic shock. The control group consisted of six female patients scheduled for surgery, in whom metronidazole was used for antimicrobial prophylaxis. Microdialysis perfusion rate was 2 μl/min. Metronidazole 500 mg was given by i/v infusion over 10 min. During the first three hours microdialysis and blood sampling were performed after every 30 min and then after each hour up to ten hours after drug administration. Metronidazole determination was performed using HPLC with detection of ultraviolet absorption at 318 nm. The groups were not comparable in terms of gender, weight and height. Metronidazole peak concentration in the plasma of septic patients was 11.4 ± 2.0 mg/l and in the plasma of the patients of the control group 16.5 ± 4.6 mg/l (p <0.05). Metronidazole peak concentration in the muscle of the septic patients was 8.2 ± 4.5 mg/l and in the muscle of the patients of the control group 7.8 ± 1.5 mg/l. Cmax/MIC90 and T >MIC90 were 23.3 ± 5.4 and 36.2 ± 25.2 hours for the septic patients and 31.1 ± 6.2 and 32.7 ± 18.1 hours for the control patients. During the years 1999–2002, MIC90 for the Bacteroides fragilis group was 0.25 ± 0.26 mg/l. The i/v administration of 500 mg metronidazole provides a concentration in the target tissue which is sufficient for killing the bacteria of the Bacteroides fragilis group.