Fluoride varnishes, both the first and the second generations, are effective in inhibiting caries, especially in children and adolescents, by reducing it on average by 43% for permanent teeth and 37% for deciduous teeth. The aim of this study was to evaluate the dynamics of in vitro fluoride ion release from first- (Duraphat) and second-generation (MI Varnish and Embrace Varnish) fluoride varnishes and the impact of the type of varnish, the time from its application and the pH of the environment on this process. Materials and methods: The test material (90 specimens), prepared from extracted human teeth, were divided into nine groups of 10 specimens each. Measured amounts of the examined varnishes were applied onto specimens and the levels of fluoride release were assessed at the baseline and after 1, 2, 24, 48 and 168 h from the application with the use of an ion-specific electrode. The specimens were immersed into artificial saliva with pH adjusted to 4, 5 and 7. The highest cumulative release of fluoride was obtained by MI Varnish (11.52 ppm/mg), regardless of the pH of the environment, whereas the lowest released fluoride concentration was achieved by Embrace Varnish (4.82 ppm/mg). In the acidic environment, the release of fluoride was significantly higher than in the neutral environment for all investigated varnishes, with no change in the overall fluoride release profile and with maximum fluoride release in the first two hours after application. The findings of this study indicate that all examined fluoride varnishes released the maximum amount of fluoride within the first hours after application and that it was related to the acidity of the immersion medium.