The expression of heat shock protein (Hsp) 25 during odontogenesis in the dental pulp and enamel organ of rat incisors was investigated by immunocytochemistry and confocal microscopy. In the process of dentin formation, immature odontoblasts first exhibited Hsp 25-immunoreactivity, and increased in immunointensity with the advance of their differentiation. In the dental pulp, in contrast, intense immunoreaction in the mesenchymal cells became weak or negative in parallel with the progress of cell differentiation. The immunoreaction for Hsp 25 in the enamel organ revealed a characteristic stage-related alteration during amelogenesis. In secretory ameloblasts, the immunoreaction for Hsp 25 was found throughout their cell bodies, intense reactivity being located near the proximal and distal terminal webs. At the maturation stage, ruffle-ended ameloblasts (RA) consistently showed Hsp 25-immunoreactivity throughout the cell bodies, whereas smooth-ended ameloblasts (SA) lacking a ruffled border were weak in immunoreaction at the distal cytoplasm. Other cellular elements of the enamel organ were negative. The subcellular localization of Hsp 25-immunoreactivity in this study appeared essentially identical to that of actin filaments as demonstrated by confocal microscopy using rhodamine-labeled phalloidin. These immunocytochemical data suggest that the Hsp 25 molecule is involved in reinforcement of the cell layer following cell movement during odontogenesis and in the formation and maintenance of the ruffled border of RA.