Background/aims: The aim of this study was to evaluate the composition of the microbiota of primary endodontic infections in I I I selected cases of single-rooted teeth with necrotic pulp. Methods: Samples were collected from the root canals using #15 Hedstroen-type files and two sterile paper points, which were introduced 1 mm short of the apical foramen. The presence, levels, and proportions of 40 different bacterial species in each sample were determined using DNA probes and checkerboard DNA-DNA hybridization techniques. Results: The mean number of species per sample was 22. Enterococcus faecalis (89.3%), Campylobacter gracilis (89.3%), Leptotrichia buccalis (89.3%), Neisseria mucosa (87.5%), Prevotella melaninogenica (86.6%), Fusobacterium nucleatum ssp. vincentii (85.7%), Eubacterium saburreum (75.9%), Streptococcus anginosus (75%), and Veillonella parvula (74.1%) were the most prevalent species. The species found in highest mean counts (over 105) were F nucleatum ssp. vincentii (13.14 x 105), E. saburreum (5.67 x 10(5)) E. faecalis (5.38 x 10(5)), N. mucosa (4.19 x 10(5)), V parvula (3.63 x 10(5)), C, gracilis (3.46 x 10(5)), Treponema socranskii (3.34 x 10(5)), Porphyromonas endodontalis (2.96 x 10(5)) Porphyromonas gingivalis (2.85 x 10(5)), Micromonas micros (2.81 x 10(5)), Prevotella nigrescens (2.68 x 10(5)) and Fusobacterium nucleatum ssp. nucleation (2.64 x 10(5)). Most of these species were also found in high proportions Conclusions: Our results suggest that several bacterial species considered to be oral pathogens seem to be implicated in the etiology of primary endodontic infections.