The purpose of this study was twofold: first, to determine the influence on the healing of the periapical tissues when selected bacterial strains and combinations thereof remain after root canal treatment; and, second, the relationship to healing of the quality of the root filling. In eight monkeys, 175 root canals, previously infected with combinations of four or five bacterial strains and with radiographically verified apical periodontitis, were endodontically treated, bacteriologically controlled, and permanently obturated. After 2–2.5 yr, the periapical regions were radiographically and histologically examined. Of these teeth, 48 root canals were also examined for bacteria remaining after removal of the root fillings. When bacteria remained after the endodontic treatment, 79% of the root canals showed non-healed periapical lesions, compared with 28% where no bacteria were found. Combinations of residual bacterial species were more frequently related to non-healed lesions than were single strains. When no bacteria remained, healing occurred independently of the quality of the root filling. In contrast, when bacteria remained, there was a greater correlation with non-healing in poor-quality root fillings than in technically well-performed fillings. In root canals where bacteria were found after removal of the root filling, 97% had not healed, compared with 18% for those root canals with no bacteria detected. The present study demonstrates the importance of obtaining a bacteria-free root canal system before permanent root filling in order to achieve optimal healing conditions for the periapical tissues.