In comparing and evaluating the instruments that are used in root debridement, roughness constitutes a standard variable that is assessed. The purpose of this study was to describe the conditions and requirements for the three-dimensional roughness measurements of tooth roots using a laser profilometer. Impressions were made of 60 instrumented and 12 untreated root surfaces, which were then measured using a dynamically focussing optical profilometer (Mikrofocus, UBM, Karlsruhe, Germany). To separate roughness from the form of the tooth, a low pass filter was applied. This meant that the longer wavelengths - which roughly approximated the root forms - were subtracted from the measured profile. We then used an individualized Fourier transformation to define the threshold at which roughness becomes waviness. Roughness parameters were Ra (average roughness) and Rz (average roughness in the z dimension) for two- and three-dimensional measurements. To describe the requirements for the measurement, we studied the effect of pixel density and the field size on the average roughness value, Ra. We found that Ra increases with pixel density until 400 per mm is reached, after which it does not change meaningfully. Furthermore, Ra is highly dependent upon the area of the field to be scanned, increasing in line with the area, and does not approach one value within the surface available on one tooth. The correlation coefficients between the two-dimensional and three-dimensional Ra and Rz values ranged from 0.7 to 0.8. We conclude that roughness values are strongly dependent on the measurement conditions and the results of one study cannot be directly compared to another. In addition, it was found that two-dimensional measurements are sufficient for characterizing root surfaces.