The aim of this study was to evaluate if the presence of supragingival plaque can influence measurements of gingival crevicular fluid flow (CFF) taken with the Periotron 6000 unit. Sixty-seven experimental sites and 67 contralateral control sites were randomly chosen. Crevicular fluid was collected on a filter paper strip for 15 seconds and the CFF was measured using the Periotron 6000 unit: CFF1. The supragingival plaque was removed from the experimental site with a Gracey curette and the plaque index (PI) was estimated. Five minutes later, a second CFF measurement was taken: CFF2. On the control tooth, the plaque was not removed, and the PI was established after the second CFF measurement. There was no significant difference between the CFF1 and the CFF2 values for the control teeth. In the experimental teeth, a PI of 3.0 increased the CFF1 (pre-plaque removal) compared to the CFF2 (post-plaque removal) value by 20.6 Periotron units (PU) (p = 0.0004). A PI of 2.0 caused a 7.3 PU increase (p = 0.0370). There were no significant changes for a PI of 1.0 or 0. In this type of experimental protocol, the results also indicated that CFF differences were not affected by the gingival index (GI) for either the experimental or control groups. These results indicate that large amounts of supragingival plaque can significantly elevate CFF measurements, as determined by the Periotron 6000 unit.