The aim of this study was to observe the potential of a mixture of ciprofloxacin, metronidazole and minocycline to kill bacteria in the deep layers of root canal dentine in situ. After the crowns of extracted teeth had been removed, the drug combination (0.5 mg of each drug), or sterile saline, as the control, was placed in the root canals which had been previously irrigated ultrasonically with G4M EDTA. The penetration and bactericidal efficacy were estimated by various procedures as follows. (1) A cell suspension of E. coli was placed into small cavities prepared parallel to the root canals on the cut planes of nine single-rooted teeth. The teeth were then entirely covered with blue inlay wax. At time 0, and at 5h, 24h and 48h after the drug combination had been applied, cells of E. coli were recovered from the cavities by washing the cavities several times with sterile saline solution, and were cultured on the surfaces of heart-infusion (HI) agar plates. Total colony-forming units were tuen counted. Bacterial recoveries decreased with time, and no bacteria were recovered 48 h after application of the drug combination, while bacteria survived in all cases with the controls, (2) After the drug combination or sterile saline had been placed into and sealed in the root canal with blue inlay wax, the teeth were placed into HI agar plates where cells of E. coli had been inoculated. After culturing, a clear zone caused by the inhibition of bacterial growth was observed around the teeth, but not in the control experiment. (3) After sampling infected root dentine of 12 freshly extracted teeth as positive controls, the drug combination (0.5 mg each) was placed in the root canals. No bacteria were recovered from the infected dentine of the root canal wall 24 h after application of the drug combination, except in one case in which a few bacteria were recovered. On the basis of these results, penetration through dentine and antibacterial efficacy of the drug combination can be expected against bacteria infecting the dentine of the root canal wall in situ when the drugs were placed in root canals which had been irrigated ultrasonically.