The role of maturation stage ameloblasts is not clear yet. The aim of this study was to verify to which extent enamel mineralizes in the absence of these cells. Maturation stage ameloblasts and adjacent dental follicle cells from rat lower incisors were surgically removed and the limits of this removal were marked by notches made in the enamel. Histological analysis confirmed that the ameloblasts had been removed within the limits of the notches. The teeth erupted and when the notches appeared in the mouth, the enamel in the experimental teeth was hard but whitish compared to the yellowish colour of the contralateral incisors used as control. SEM images revealed similar enamel rod arrangement in both groups. Decreased mineral content was observed in some specimens by polarized light microscopy, and microhardness values were much lower in the experimental teeth. FTIR analysis showed that higher amounts of protein were found in most experimental teeth, compared with the control teeth. Enamel proteins could not be resolved on 15% SDS-PAGE gels, suggesting that most of them were below 5kDa. These results suggest that the enamel matured in the absence of ameloblasts has increased protein content and a much lower mineral content, suggesting that maturation stage ameloblasts are essential for proper enamel mineralization.