An autoradiographic study using 55Fe was performed to investigate the distribution of iron in the ameloblasts and enamel of rat incisors. The study of methodological problems of 55Fe autoradiography revealed that in the undecalcified sections, the radiation by 55Fe within the ameloblasts and pigmented enamel produced grain scattering over the facing enamel surface and ameloblasts, respectively. The findings indicate that the autoradiographic observation of the enamel surface should be made using the incisors freed from the enamel organ and that the observation of the ameloblasts should be done in the region other than the zone of enamel pigmentation. After the 55Fe injection, 55Fe incorporation into the ameloblasts was observed in the maturation stage, reaching the maximum at 24 hours. The peak of 55Fe distribution in the ameloblasts moved from the early stage to the later stage of maturation between 24 hours and 7 days after the injection. At 3 and 7 days after the 55Fe injection, 55Fe was deposited markedly on the enamel surface at the end of maturation where the enamel pigmentation occurs. The present study established the utility of 55Fe autoradiography when applied on the undecalcified section of rat incisors and it was demonstrated that iron is incorporated into the maturation ameloblasts and secreted onto the enamel surface at the end of maturation.