The microbiota of root caries lesions of different grades of severity were studied. Fourteen lesions were examined. The experimental design of the study allowed correlation of histopathologically distinguishable stages with specific and distinct microbial populations. Dentin samples were ground in a sterile mortar and cultured anaerobically on nonselective Columbia blood agar plates supplemented with 5% hemolyzed human blood and on media selective for Lactobacillus spp. and streptococci. The cultivable microbiota were quantitatively speciated using Rapid ID 32A, Rapid ID 32 Strep, API 20 Strep, API ZYM, and API 50 CH tests and SDS-PAG electrophoresis. In initial as well as in advanced lesions gram-positive bacteria accounted for approximately 90% of the CFUt. The proportion of Actinomyces, and in particular A. naeslundii was significantly higher (p < 0.05) in initial lesions than in advanced lesions. In contrast, the percentage of Streptococcus and especially S. mutans was higher (p < 0.05) in advanced than in initial lesions. Surprisingly low (0.8% of the CFUt) was the percentage of lactobacilli in advanced lesions. Gram-negative bacteria formed a minor part of the microbiota in both initial and advanced lesions. Among the gram-negative isolates, Prevotella, Selenomonas, and Bacteroides spp. were most noticeable. In advanced lesions, only the outermost layer of 0.5 mm thickness was populated by a high number of bacteria; the following segments harbored a negligible number of bacteria only. It is concluded that root caries is a continuous destruction process which is restricted to a subsurface zone of limited depth. The necrotic dentin is successively worn away, leading to a saucer-shaped cavitation which is repopulated by plaque. The creation of cavitations favors an aciduric flora. This might explain the succession of bacterial populations observed during the destruction process.