The lower incisors of young rats were dissected, immersed in physiological saline containing 45Ca under various conditions, and processed for autoradiography. The data were compared with those from in vivo 45Ca autoradiography. In secretory-stage enamel, wiped free of the enamel organ and immediately immersed in radioactive saline, there was intense labelling in the surface layers. The labelled area expanded only gradually into the deeper layers at a rate similar to that observed in vivo. Labelling in the enamel was similar in pattern but much weaker in intensity when the incisor was identically treated in vitro with the enamel organ attached. Glutaraldehyde pretreatment of the exposed enamel abolished expansion of the labelled area, whereas a hypochlorite pretreatment allowed a rapid diffusion of the isotope into the deeper layers of the secretory-stage enamel. The findings confirm the role of the enamel organ as a diffusion barrier to the penetration of calcium from the extracellular fluid to the secretory-stage enamel, and suggest an intimate correlation between physicochemical properties of the organic enamel matrix and the rate of surface-to-interior diffusion of calcium within the secretory-stage enamel of rat incisors.