In this study, 120 mg of micronomicin (MCR) was given to 15 cases intended for cholecystectomy intramuscularly by a single injection or 5 consecutive injections (in the evening of day -2, morning and evening of day -1, morning of day 0, and 1 hour before operation) or intravenously by 1-hour drip infusion, and levels of MCR in serum, B bile and gallbladder tissues were determined by means of HPLC and bioassay. The serum level of MCR 30 minutes after consecutive injections (8 cases) was 11.86 +/- 1.90 micrograms/ml, significantly higher than that after the single injection, 7.08 +/- 0.93 micrograms/ml. The highest bile level of MCR after consecutive injections was 10.0 micrograms/ml. The average level in 4 detectable cases, 6.33 +/- 2.06 micrograms/ml, came up to 50% of the serum level and was higher than that after the single injection, 3.53 +/- 1.39 micrograms/ml. The gallbladder tissue level of MCR after consecutive injections was 4.5 micrograms/g at the highest and 2.51 +/- 0.73 micrograms/g on the average in 5 detectable cases. This was equivalent to 20% of the serum level and higher than that after the single injection, 1.63 +/- 0.26 micrograms/g. The MIC of MCR could be determined against 8 of 10 strains detected in B bile. Against E. coli and K. pneumoniae, main causal bacteria of bile duct infections, it was as low as 0.39 to 0.78 micrograms/ml. Levels of MCR in bile and gallbladder tissues determined in this study exceeded by far the above MIC. From these results, it can be expected that clinical administration of MCR at 2 doses of 120 mg daily for 3 days or more will give rise to a sufficiently antibacterial effect against Gram-negative bacilli.