Objective: To investigate the effects of different background colours (black, white or pink), direction and intensity of ambient light, measuring position, and the adjacent teeth, on the in vitro colour measurement of maxillary anterior teeth, using the Minolta CR-321 colorimeter. Methods: Ten extracted human maxillary central incisors were selected. A fibre-optic light MI-150 was used as the ambient illuminant. Teeth were irradiated from a 3- or 12-o'clock direction. L*a*b* values of seven sites on the labial surfaces were obtained by means of the Minolta CR-321 colorimeter, using three background colours, with or without the adjacent teeth. The recorded data were analysed with two-tailed Student t tests and analysis of variance (α = 0.05). Results: The ambient light did not affect the colour measurement of anterior teeth, regardless of the presence or absence of the adjacent teeth. There were no statistically significant differences in L*a*b* values at the same position under different background colours, except ΔE12 (colour difference between site 1 and site 2) between black and white backgrounds. ΔE12 (under black background), ΔE13 and ΔE15 were greater than 1.5, while the others were lower than 1.5. Conclusion: The background, ambient light and the presence of adjacent teeth did not affect the colour measurement of anterior teeth using the Minolta CR-321 colorimeter in vitro. The inherent disadvantages of using the naked eye during clinical visual shade assessment may be overcome by the colorimeter. Keywords: background colour, colorimeter, colour measurement, in vitro, tooth colour