The aim of the present study was to calibrate the Periotron® model 8010 with volumes of three different fluids (distilled water, serum, and saliva) and to identify which of the three is the most reliable, feasible, and reproducible for routine calibration. A total of 450 samples of Periopaper® were divided into three groups (150 each per group): distilled water, serum matrix and saliva. A calibration curve was run with 0.25, 0.50, 0.75, 1.00 and 1.25 µl of each of the fluids, and the results were determined in Periotron units (PU). Statistical analysis was performed with one-way ANOVA followed by Bonferroni's post hoc test and a linear equation. Distilled water presented the lowest levels of PU at all volumes, while serum showed the highest levels at high volumes. Linear regression equations rendered similar slopes for saliva and distilled water, while serum was statistically different. Saliva presented a reproduction percentage of 99.7%, which indicated better accuracy and precision than serum and distilled water. Saliva is more reliable and accurate than water or serum for the purpose of calibration of the Periotron® model 8010, though it shares drawbacks with serum. Distilled water is more easily available and does not require any additional procedure, in addition to producing a similar slope to saliva and a smaller deviation from the media than serum.