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The purpose of this study was to investigate the influence of the color of positioning guides on the CIEL*a*b* color coordinates of ceramic, resin composite and bovine tooth surfaces measured by a clinical spectrophotometer. Positioning guides (n=10) in different colors (translucent, purple, yellow, green and, blue) were made for each surface (ceramic, resin composite and bovine tooth). The CIEL*a*b* color coordinates were measured with the positioning guides and with no positioning guide (control group). The ΔL*, Δa*, Δb*, ΔE*ab, and ΔE00 were calculated between the control group and the different groups. The CIEL*a*b* color coordinates were analyzed by one-way ANOVA followed by the post-hoc Tukey test (α=0.05). Color differences were interpreted using the 50:50% perceptibility and acceptability visual thresholds. Positioning guides with different colors presented significant differences for all surfaces and color coordinates. ΔE*ab, and ΔE00 mean values for all surfaces and positioning guide colors exceeded the acceptability visual thresholds, except for the bovine tooth surface measured with the green positioning guide. The shade measurement was affected by the color of the positioning guides regardless of the surface that was evaluated.