The aim of the study was to measure the fluoride (F) concentration in plaque after a single topical application of different fluoride varnishes with contrasting levels of F. Thirty adolescents (12-17 years) with fixed orthodontic appliances were randomly assigned to one of three groups: Bifluoride (6% F), Duraphat (2.23% F) and Fluor Protector (0.1% F). The varnishes were applied after professional cleaning in one upper quadrant, leaving the opposite quadrant untreated according to the split-mouth technique. Pooled plaque samples from each quadrant were collected at baseline and 3 days, 7 days and 30 days after the varnish treatment, and fluoride was analysed by microdiffusion. All fluoride varnishes increased the fluoride concentration in plaque compared with baseline, and the mean values varied between 23 and 138 ng F/mg after 3 days, depending on varnish F concentration. Compared with the control quadrant, statistically significant elevations were recorded for Bifluoride after 3 days and 7 days and Duraphat after 3 days, while no significant differences were revealed in the Fluor Protector group. The fluoride concentration in plaque was back to baseline levels for all participants in the Duraphat group after 7 days, while some individuals in the Bifluoride and Fluor Protector groups still registered slightly increased levels after 30 days. The results suggest that fluoride varnish treatments resulted in elevated fluoride levels in plaque adjacent to fixed orthodontic appliances for a period of up to 1 week, although different patterns was disclosed for the various brands.