The Periotron 6000® fluid analyser has become widely used as a diagnostic tool for a variety of oral diseases and recent work has questioned its reliability. This paper investigates for the first time, the detailed calibration curves of 2 Periotron 6000 machines across a range of 23 different fluid volumes. Within and between machine reliability is analyzed and the shape of the calibration line determined. The measurement errors incurred by using a single fluid sample, as opposed to mean values of triplicate samples are also determined. We conclude that there are 3 sections to the calibration line, 2 linear and a quadrilateral zone, and that 2 separate regression equations should be used; from 0–0.1 μl and from 0.1–1.0 μl. Within machine calibration errors were only 3.2±7.5%, but values for volumes below 0.2 μl were as high as 18.7%. Using a single fluid sample rather than mean values of multiple samples, incurred a further 4±4% error, which was as high as 7% for volumes lower than 0.12 μl. Whilst significant differences in volume reading existed between different machines (p<0.0004) and between the same volumes of different fluids (p<0.00001), individual Periotron calibrations were extremely reproducible and reliable. We conclude that the Periotron 6000 is a reliable and convenient instrument for measuring fluid volumes greater than 0.2 μl. For volumes lower than 0.2 μl errors in measurement may be too high for some investigations, but this is likely to be due to problems with evaporation and with measurement technique, rather than errors directly due to the Periotron itself. Finally, for optimum accuracy, the digital display should be re‐set to zero after each samp