Objectives: The aim of this longitudinal, controlled study was to determine factors associated with mutans streptococci infection and development of caries in a group of preschoolers. Methods: At commencement of study, 63 caries-free subjects who were recruited since birth were divided into a study group of 24 children who were colonized with mutans streptococci, and a control group of 39 children without the bacteria. The children were recalled every 3 months from mean age of 33 mths to mean age of 55 mths. Results: Mutans streptococci infection was associated with visible plaque (p<0.01), enamel hypoplasia (p<0.05), commencement of toothbrushing after 12 mths of age (p<0.05), lack of parental assistance with toothbrushing (p<0.025), and increased number of hours of childcare (p<0.05). Of the 20 children who became infected with mutans streptococci during the study, 4 (20%) were colonized at a mean age 21-36 mths, 9 (45%) at mean age of 37-48 mths, and 7 (35%) at mean age of 49-72 mths (p<0.001). Eight children who developed caries had greater numbers of hypoplastic teeth (p<0.001), ate sugar containing snacks (p<0.05), and brushed less frequently (p<0.025) compared to those who remained caries free. In contrast, more children who remained caries-free brushed regularly with 0.2% chlorhexidine gel (p<0.01). Conclusions: Lack of oral hygiene, consumption of sugar-containing snacks and enamel hypoplasia are significant risk factors for mutans streptococci infection and caries initiation in preschool children. Daily brushing with 0.2% chlorhexidine gel can prevent caries in preschoolers. This study was funded by National Health and Medical Research Council of Australia and the Australian Dental Research Foundation.