Laser fluorescence (LF) and differential reflectometry (DR) are two new optical methods which have been used to help diagnose subgingival deposits of dental calculus. This study compared the performance of LF using the KEY3 laser system versus DR using the DetecTar system under controlled laboratory conditions designed to simulate clinical conditions as much as possible. A total of 30 extracted human posterior teeth were set in an anatomical configuration in stone typodonts with impression material replicating the periodontal soft tissues. LF was more accurate than DR (76.2% vs. 68.2%) and gave higher reproducibility (Bangdiwala's B statistic 0.71 vs. 0.54). The better performance of LF makes it the preferred option of the two methods for detection of subgingival dental calculus.