Objective: This randomized, single-controlled study was performed to validate in vivo the efficacy of single Fluorinex treatment by examining fluoride incorporation into enamel using electron microscopy. Method and Materials: Twenty healthy participants referred for routine dental treatment which also required extractions of at least two teeth as part of their treatment plan were included in this study. For each participant, one randomly selected tooth was extracted and sent for blind electron microscopic fluoride assay (control, C). Next, following a single Fluorinex treatment, the second (test, T) tooth was extracted and sent for the same assay. Intra-individual and intergroup fluoride content comparisons were performed, between control and test teeth. Results: Highly significant intra-individual and intergroup differences were found between the treated and untreated teeth. Fluoride atomic percent (at%) and weight percent (wt%) estimated least squares means of untreated group were found to be 0.56 and 0.48, and in the treated group 17.35 and 14.35. This increase in fluoride at% [16.78 ± 2.3 (SE)], was also significant statistically (P .0001); likewise, the increase in fluoride wt % [13.86 ± 1.97 (SE)] was similarly significant (P .0001). The system was well tolerated by the participants with minimal transitional mild side effects. Conclusion: In vivo fluoride application using the active Fluorinex system resulted in a significant increase of fluoride content in the enamel of adult permanent teeth compared to untreated internal controls. Keywords: caries prevention, fluoride, Fluorinex, fluoritray, third molars, topical fluoridation