The influence of urine on the in vitro activities of various antibiotics used in the therapy of urinary tract infections was assessed by the microbrothh dilution method. Thirty Escherichia coli strains were used in this study: ten strains susceptible to ampicillin, ten strains resistant to ampicillin and ampicillin+sulbactam and ten were extended spectrum beta-lactamase producer strains. The MICs of ampicillin, ampicillin+sulbactam, cephalothin, cefuroxime, ceftriaxone, amikacin, ciprofloxacin, sulfamethoxazole and trimethoprim were performed parallel in Mueller Hinton broth and human urine by the microbrothh dilution method. The MIC90 of all antibiotics except cephalothin were higher in the urine. MICs performed in the urine were found significantly higher than those performed in brothh media. This study demonstrated that MICs of antibiotics are influenced by the human urine and MICs of some antibiotics used in the treatment of urinary tract infections may be overestimated by the standard antibiotic testing methods.