In order to clarify the in vivo effect of the new quinolones in the urinary tract, we investigated the antimicrobial activities against Escherichia coli in the human urine. Human urine was prepared from a normal volunteer. After cations were removed by chelating resin, human urine was supplemented with 50, 100, or 500 pg of Mg2+ per ml using MgCl₂ or 10, 50, or 150μg of Ca²⁺ per ml using CaCl2. The pH of human urine was adjusted to 5.5, 7.0, or 8.0 with HC1 or NaOH. Four clinical isolates of E. coli and E. coli NIHJ JC-2 were used. And antimicrobial activities of the new quinolones (norfloxacin, enoxacin, ofloxacin and ciprofloxacin) against these strains were measured using various urines as mediums. Antimicrobial activities of the new quinolones were reduced in the presence of high concentration of magnesium or low pH of urine. However, there was no influence of calcium concentration of urine on antimicrobial activities of the new quinolones. From these results, the component of the urine should be checked in the treatment of the patients with urinary tract infection.