The aim of this study was to evaluate, by checkerboard DNA-DNA hybridization, the composition of the microbiota of primary endodontic infections in cases associated with exposed (n = 30) and unexposed (n = 30) pulp space.Samples were collected by means of a #15 H-type file and 2 sterile paper points from 60 single-rooted teeth with necrotic pulp and periapical lesions. The presence, levels, and proportions of 40 bacterial species were determined by checkerboard DNA-DNA hybridization.The species found in higher counts (×10 5 ) in exposed pulp space cases were Eubacterium saburreum, Fusobacterium nucleatum ssp. vincentii, Tannerella forsythia, Enterococcus faecalis, Neisseria mucosa, Campylobacter gracilis, and Prevotella nigrescens, and in unexposed pulp space cases they were F. nucleatum ssp. vincentii, N. mucosa, E. faecalis, E. saburreum, C. gracilis, and Porphyromonas gingivalis. Counts of F. nucleatum ssp. vincentii, Campylobacter sputigena, Capnocytophaga showae, Treponema socrenskii, Porphyromonas endodontalis, Eikenella corrodens, and Capnocytophaga ochracea were significantly higher in unexposed pulp space cases (P < .05).The data of the present investigation suggested specific differences between the composition of the microbiota in cases with exposed and unexposed pulp space and an association between higher levels of some specific species and unexposed pulp space cases.