The aim of this study was to evaluate the influence of different backgrounds on spectrophotometric colour values of natural teeth. Twenty volunteers (10 males and 10 females) with a mean age of 25 years and 9 months (±3 years and 2 months) were each subjected to 4 spectrophotometric measurements of their upper right central incisor. Each sample was measured with alternatively black, 50 % grey, white or no background (positive control). ΔE medians ranged from 0.9 to 5.9. All artificial backgrounds presented significant differences (p < 0.05) when compared to values obtained without any background. No significant differences were observed between black and 50 % grey background (p > 0.05). If an artificial background needs to be used, as for example when performing in vitro studies, preference should be given to a black background as it approaches best the clinical situation (i.e. no background). Even if no statistically significant differences were found when compared with the grey background, the black background should be preferred due to its lower ΔE medians, standard deviation as well as lower minimum and maximum values.