Summary.This study was carried out to investigate the presence and types of bacteria invading the deep layers (0.5–2.0 mm from the surface of the root canal wall) of infected dentine of human root canals by sampling with an anaerobic glove box system the split surfaces of eight freshly extracted teeth. More bacteria were recovered after incubation in an anaerobic glove box than after aerobic incubation in air with 30 per cent CO2. Out of 256predominant bacteria isolated, 205 isolates (80 per cent)were bligate anaerobes. These findings suggest that the environment of deep layers of endodontic dentmal lesions is anaerobic and favours the growth of anaerobes. Among the obligate anaerobic isolates, strains belonging toLacto‐bacillus(30 per cent) andStreptococcus(13 per cent) were predominant, followed byPropionitacterium(9 per cent). No strains of obligate anaerobic Gram‐negative rods were isolated. The tnicroflora of deep layers of infected root dentine is somewhat similar to that of the deep layers of carious lesions in coronal de