Since in vitro and animal studies suggest that the combination of starch with sucrose may be more cariogenic than sucrose alone, the study assessed in situ the effects of this association applied in vitro on the acidogenicity, biochemical and microbiological composition of dental biofilm, as well as on enamel demineralization. During two phases of 14 d each, fifteen volunteers wore palatal appliances containing blocks of human deciduous enamel, which were extra-orally submitted to four groups of treatments: water (negative control, T1); 2 % starch (T2); 10 % sucrose (T3); and 2 % starch þ 10 % sucrose (T4). The solutions were dripped onto the blocks eight times per day. The biofilm formed on the blocks was analysed with regard to amylase activity, acidogenicity, and biochemical and microbiological composition. Demineralization was determined on enamel by cross-sectional microhardness. The greatest mineral loss was observed for the association starch þ sucrose (P,0·05). Also, this association resulted in the highest lactobacillus count in the biofilm formed (P,0·05). In conclusion, the findings suggest that a small amount of added starch increases the cariogenic potential of sucrose. Among dietary carbohydrates, starch has been pointed out as noncariogenic or slightly cariogenic when used as the sole source of carbohydrate in the diet. This observation has been supported by experiments on dental biofilm acidogenicity (Stephan, 1940;Imfeld, 1977;Lingström et al. 1989), experimental studies with animals (König & Grenby, 1965;Green & Hartles, 1967;Hefti & Schmid, 1979;Bowen et al. 1980), controlled studies in man (Gustaffson et al. 1954), epidemiological data (Marthaler & Froesch, 1967;Fisher, 1968;Newbrun et al. 1980) and in situ experiments (Lingström et al. 1994), which demonstrated that starch is less cariogenic than sucrose.However, while in primitive diets starch was consumed as the main energy source, in contemporary ones it is consumed simultaneously or interspersed with sucrose (Lingström et al. 2000). This combination, which is consumed by both adults and children, may influence dental biofilm composition and consequently dental caries. Thus, a greater prevalence of caries lesions was found in children who consume milk supplemented with a combination of cereal and sucrose (Mattos-Graner et al. 1998). Such observation in human subjects is supported by the results of experimental caries studies in animals (Firestone et al. 1982;Mundorff-Shrestha et al. 1994), suggesting that starch would enhance the cariogenic potential of sucrose.The explanation for the greater cariogenicity of the association of dietary starch with sucrose may reside in the dental biofilm formed. It is well known that the biofilm formed in the presence of sucrose is more cariogenic due to its high concentration of extracellular insoluble polysaccharides (IP), which alter the matrix of the biofilm, making it more porous (Dibdin & Shellis, 1988). These polysaccharides are produced from sucrose by bacterial enzymes named glucosyltransferases....