Aims: To elucidate the first colonizers within in vivo dental biofilm and to establish potential population shifts that occur during the early phases of biofilm formation. Methods and Results: A 'checkerboard' DNA - DNA hybridization assay was employed to identify 40 different bacterial strains. Dental biofilm samples were collected from 15 healthy subjects, 0, 2, 4 and 6 h after tooth cleaning and the composition of these samples was compared with that of whole saliva collected from the same individuals. The bacterial distribution in biofilm samples was distinct from that in saliva, confirming the selectivity of the adhesion process. In the very early stages, the predominant tooth colonizers were found to be Actinomyces species. The relative proportion of streptococci, in particular Streptococcus mitis and S. oralis, increased at the expense of Actinomyces species between 2 and 6 h while the absolute level of Actinomyces remained unaltered. Periodontal pathogens such as Tannerella forsythensis ( Bacteroides forsythus), Porphyromonas gingivalis and Treponema denticola as well as Actinobacillus actinomycetemcomitans were present in extremely low levels at all the examined time intervals in this healthy group of subjects. Conclusion: The data provide a detailed insight into the bacterial population shifts occurring within the first few hours of biofilm formation and show that the early colonizers of the tooth surface predominantly consist of beneficial micro-organisms. Significance and Impact of the Study: The early colonizers of dental plaque are of great importance in the succession stages of biofilm formation and its overall effect on the oral health of the host.