The distribution and prevalence of Streptococcus mutans and Streptococcus sobrinus were determined at three sub-sites in human approximal plaque: away from (A) to the side of (S) and below (B) the contact area. Small plaque samples were taken from all three sub-sites on clinically sound approximal surfaces of a single premolar from each of 21 schoolchildren. S. mutans was detected significantly more often and in higher proportions than S. sobrinus from sub-sites A (p = 0.019), S (p = 0.034) and B (p = 0.004). S. mutans was detected in highest proportions from the B site compared to the A site (p = 0.025); there were no significant differences in the isolation frequency or prevalence of S. sobrinus from any of the sub-sites. S. mutans and S. sobrinus were never isolated together from the A sub-sites and were recovered together most commonly from the B sub-sites (p < 0.01). It is concluded that S. mutans and S. sobrinus preferentially colonise the most caries-prone site apical to the contact area.