Amino acid transporters play important roles in supplying nutrients to cells. In our current study, we investigated the expression of LAT1 and measured the amino acid uptake in ameloblast cultures to further elucidate the roles of this transporter during the differentiation of these cells. RT-PCR, observations of cell morphology, Alizaline red-S staining, and uptake analyses were performed following the experimental induction of differentiation in the cultures. LAT1 mRNA was detectable and found to gradually increase over time whereas LAT2 mRNA was not evident in the ameloblast cultures. Transcripts of 4F2hc, a cofactor of LAT1 and LAT2, were also found to be expressed in ameloblast cultures and increase with time. Amelogenin mRNA was expressed in the early stage ameloblast cultures. L-leucine uptake was observed to increase over 14 days of growth in culture. Our data suggest that LAT1 has a key role in the differentiation of ameloblasts and in providing these cells with neutral amino acids, including several essential amino acids.