Objective: The aim of this study was to examine the calbindin D-28k immunoreactivity in carious teeth to know whether this protein may have a function in tertiary dentine formation. Methods: Human extracted teeth with or without carious lesions were immersion-fixed with Zamboni fixative, demineralized in 4.13% EDTA solution (pH 7.4), frozen-sectioned, and processed for calbindin immunoreactivity and hematoxylin-eosin stain. The intensity of the immunostaining was evaluated by quantitative densitometry. Results: In intact teeth, numerous odontoblasts were aligned underneath the secondary dentine and their cell bodies showed the immunoreactivity. In carious teeth, tertiary dentine had poor- or rich tubular patterns under the carious lesion. Underneath the tubule-poor tertiary dentine, distinct odontoblasts could not be seen at the central site. However, some cells with a flat appearance were located at this site and were immunonegative for calbindin D-28k. On the other hand, columnar odontoblasts were seen at the peripheral site, and their cell bodies and processes showed strong immunoreactivity. Underneath the tubule-rich tertiary dentine, columnar odontoblasts were abundantly distributed, and the strong immunoreactivity was observed in their cell bodies and processes. The immunoreactivity in odontoblasts underneath the tertiary dentine with poor or rich tubular pattern was more intense than that for the secondary dentine in intact teeth (P < 0.05). On the other hand, the intensity of the immunoreactivity in odontobtasts was similar underneath the secondary dentine in intact and carious teeth. Conclusions: The present study demonstrated that calbidin D-28k was actively synthesised by odontobtasts under the carious lesion. These findings may suggest that this protein plays an important rote in the tertiary dentine formation. (C) 2003 Elsevier Ltd. All rights reserved.