An in vitro model for dentinal tubule infection of root canals was developed. Cylindrical dentin specimens, 4 mm high with a diameter of 6 mm and a canal 2.3 mm wide, were prepared from freshly extracted bovine incisors. The cementum was removed from all dentin blocks. The tubules were opened by four-minute treatments with 17% EDTA and 5.25% NaOCI before being infected with Enterococcus faecalis ATCC 29212 in yeast extract-glucose broth. Bacteria rapidly invaded the tubules. After three weeks of incubation, a heavy infection was found 400 μm from the canal lumen, and the front of the infection reached 1000 μm in some blocks. Camphorated paramonochlorophenol (CMCP) and a calcium hydroxide compound, Calasept®, were tested for their disinfecting efficacy toward E. faecalis- infected dentin. Liquid CMCP rapidly and completely disinfected the dentinal tubules, whereas CMCP in gaseous form disinfected tubules less rapidly. Calasept® failed to eliminate, even superficially, E. faecalis in the tubules. The method used in bacteriological sampling allowed for sequential removal of 100-μm-thick zones of dentin from the central canal toward the periphery. Control specimens were uniformly in fected and yielded growth in bur samples up to some 500 μm from the surface. The model proved quite sensitive and seems suitable for in vitro testing of root canal medicaments.