The aim of the study was to evaluate the precision of a reflectance spectrophotometer during longitudinal assessment of tooth colour in vivo. The spectrophotometric data of five standardized circular areas on the labial surfaces of six teeth (four upper and two lower), from 22 dental students, were recorded on three separate days (1st, 3rd and 30th) employing a repeated-measuring design (n = 3). Total colour differences were calculated according to the equation DeltaEpsilon = [(DeltaL*)(2) + (Deltaa*)(2) + (Deltab*)(2)](1/2). It was found that all measured teeth areas, recorded by the same examiner (DeltaEpsilon(INTRA)), demonstrated minor colour changes during the three different time intervals, which did not exceed in every case the 50:50% perceptibility threshold of DeltaEpsilon = 1, thus indicating a good match. The smallest DeltaEpsilon values were recorded for the upper central incisors, whereas lower central incisors and upper first premolars revealed the most significant colour differences (0.68 +/- 0.21 and 0.70 +/- 0.25 DeltaEpsilon-units, respectively). The central zone of the middle third of each labial tooth surface exhibited the most precise recordings. With regard to interexaminer reproducibility (DeltaEpsilon(INTER)), the mesial and distal areas of all measured teeth presented the most statistically significant colour differences (0.48 +/- 0.15 and 0.50 +/- 0.17 DeltaEpsilon-units, respectively). It is concluded that the spectrophotometer tested in the present study can provide a precise measurement of tooth colour in vivo. However, in rejection of the null hypothesis, the type (posterior teeth, mandibular incisors) and the mesial and distal areas of natural teeth affected the repeatability and reproducibility of intraoral spectrophotometric measurements.