Based on numerous microbiological studies performed over the past several decades, it is clear that mutans streptococci can cause human root caries. S. mutans fulfills the criteria for implicating bacteria in the etiology of a mixed infection. For example, S. mutans is found in high numbers in lesion sites, higher than on sound root surfaces in the same subject. Subjects make elevated antibody levels to S. mutans antigens. The organism produces a number of virulence factors including metabolic acid from dietary sucrose and extracellular polysaccharides which facilitate bacterial colonization of tooth surfaces. Eliminating or reducing the number of S. mutans reduces the number of root caries lesions and can even result in "healing" of incipient lesions. There is also data demonstrating the cariogenic potential of S. mutans in animal models. Clearly, S. mutans fulfills the aforementioned requirements. Further, there is also evidence to implicate Lactobacillus as being important in the pathogenesis of root caries by virtue of its association with S. mutans in these lesions. There is less recent evidence regarding the importance of Actinomyces in this disease. While this microorganism is present in root caries and while animal studies clearly point to their cariogenic potential, more recent studies with few exceptions fail to find much association between Actinomyces and root caries. There is an important caveat, however. The Actinomyces may have subspecies groups which are more highly virulent and more closely involved in the etiology of root caries than other groups. For example, A. viscosus serovar 2 is associated with root caries. This and other subspecies groups may produce certain virulence factors not found within Actinomyces species as a whole. For this group of microorganisms and for other potential pathogens, techniques in molecular biology such as 16S ribosomal RNA sequencing offers the hope of more precisely defining species and unraveling what may be largely problems in bacterial taxonomy. Ribosomal RNA sequencing may reveal taxonomic relationships not apparent with classical phenotypic or serologic analyses. Other molecular methods, such as DNA or RNA probes to specific virulence factors may also reveal relationships between clinical lesions and microorganisms possessing these virulence factors. Finally, there are clearly a number of additional species which may have importance in root surface caries as shown in some studies. These techniques can be used to identify the distribution of novel, even uncultivable bacteria in root caries lesions and in this way establish their role in this important disease.