Transmission of Streptococcus mutans, a major dental caries pathogen, occurs mainly during the first 2.5 years of age. Children appear to acquire S. mutans mostly from their mothers, but few studies have investigated non-familial sources of S. mutans transmission. This study prospectively analysed initial S. mutans oral colonization in 119 children from nursery schools during a 1.5-year period and tracked the transmission from child to child, day-care caregiver to child and mother to child. Children were examined at baseline, when they were 5-13 months of age, and at 6-month intervals for determination of oral levels of S. mutans and development of caries lesions. Levels of S. mutans were also determined in caregivers and mothers. A total of 1392 S. mutans isolates (obtained from children, caregivers and mothers) were genotyped by arbitrarily primed PCR and chromosomal RFLP. Overall, 40.3 % of children were detectably colonized during the study, and levels of S. mutans were significantly associated with the development of caries lesions. Identical S. mutans genotypes were found in four nursery cohorts. No familial relationship existed in three of these cohorts, indicating horizontal transmission. Despite high oral levels of S. mutans identified in most of the caregivers, none of their genotypes matched those identified in the respective children. Only 50 % of children with high levels of S. mutans carried genotypes identified in their mothers. The results support previous evidence indicating that non-familial sources of S. mutans transmission exist, and indicate that this bacterium may be transmitted horizontally between children during the initial phases of S. mutans colonization in nursery environments.