The cariogenicity of starch alone or in combination with sucrose is controversial and the effect on dentine demineralization and on the dental biofilm formed has not been explored under controlled conditions. A crossover, single-blind study was conducted in four steps of 14 days each, during which 11 volunteers wore palatal appliance containing 10 slabs of root dentine to which the following treatments were applied extraorally: 2% starch gel-like solution (starch group); 10% sucrose solution (sucrose group); a solution containing 2% starch and 10% sucrose (starch + sucrose group), or 2% starch solution followed by 10% sucrose solution (starch] sucrose group). On the 14th day of each phase the biofilms were collected for biochemical and microbiological analyses, and dentine demineralization was assessed by hardness. A higher demineralization was found in dentine exposed to sucrose and starch sucrose combinations than to starch alone (p < 0.01), but the sucrose-containing groups did not differ significantly from each other (p > 0.05). The concentrations of soluble and insoluble extracellular polysaccharides (EPS), and the proportion of insoluble EPS, were lower in the biofilm formed in presence of starch (p < 0.01) than in those formed in the presence of sucrose or sucrose/starch combinations; however, no significant difference was observed among the groups containing sucrose (p > 0.05). RNA was successfully isolated and purified from in situ biofilms and only biofilms formed in response to sucrose and starch/sucrose combinations showed detectable levels of gtf B and gtf C mRNA. The findings suggest that the combination of starch with sucrose may not be more cariogenic to dentine than sucrose alone Ver menos