Objectives: Because subgingival dental calculus in periodontal pockets is associated in the etiopathogenesis of progressive human periodontitis, and is difficult to accurately detect with conventional manual explorers and probing instruments, there is an urgent clinical need for more reliable diagnostic methods for the detection and localization of subgingival dental calculus. A low-power ( 40). Results: A total of 50 root surfaces exhibited a modified SCI score = 0 (no root surface dental calculus detected), whereas 19 root surfaces revealed modified SCI scores = 1 (root surface dental calculus detected in thin deposits, but not in a markedly-raised ledge), and 31 root surfaces had modified SCI scores = 2 (root surface dental calculus detected in a markedly-raised ledge). A high level of both intra- and inter-examiner reproducibility of visible red laser fluorescence intensity readings was found with both tooth root evaluation protocols, despite the marked differences between the two dentist examiners in their educational backgrounds and length of clinical dental care experience, with correlation coefficient values ranging from r = 0.948 to r = 0.999 for duplicate assessments made by the two independent examiners themselves and between them. Mean visible red laser fluorescence intensity values recorded by the two independent examiners with the instrument perpendicularly directed along tooth root surfaces (first evaluation protocol) were 98.9 (standard deviation ± 0.4) and 99.0 (standard deviation ± 0.0), respectively, on dental calculus-positive root surfaces, which were significantly greater than mean values of 10.9 (standard deviation ± 6.0) and 12.3 (standard deviation ± 8.1), respectively, recorded on dental calculus-negative root surfaces (P 40 for visible red laser fluorescence intensity values offered 90% sensitivity, 100% specificity, a 100% positive predictive value, a 90.9% negative predictive value, and an odds ratio relationship of 36.6 [95% confidence interval = 16.7, 80.2] for the presence of dental calculus on tooth root surfaces. Conclusions: These in vitro findings document, for the first time, a high level of intra- and inter-examiner reproducibility of visible red laser fluorescence intensity measurements on human tooth root surfaces, regardless of the whether the instrument is directed either perpendicular or parallel to extracted tooth root surfaces. Dental calculus- positive root surfaces on extracted teeth exhibited significantly higher visible red laser fluorescence intensity scores than dental calculus-negative root surfaces, particularly when dental calculus deposits were present in markedly-raised ledges. In addition, a threshold level of > 40 for visible red laser fluorescence intensity readings offered greater diagnostic accuracy than a threshold level of ≥ 5 for identification of dental calculus on root surfaces of extracted teeth. These findings provide further in vitro validation for use of the visible red laser fluorescence-emitting instrument for detection of dental calculus on root surfaces of human teeth. Additional validation studies, conducted clinically in vivo, on the visible red laser fluorescence-emitting instrument are warranted. / Oral Biology