OBJECTIVE: This study examined the in vitro caries inhibiting potential of fluoridated and non-fluoridated rewetting agents that are applied to acid-etched enamel and dentine before the use of a water-free, dentine adhesive. MATERIALS AND METHODS: Twelve caries-free premolars were divided into three groups of four teeth each. 2 x 3 x 1.5 mm cavities were prepared on the mesial and distal surfaces of each tooth, with half of the cavosurface margin in enamel and half in root dentine. In Group I (control), One-Step (Bisco, Schaumburg, USA) was applied without etching or rewetting agents. In Group II, cavities were acid-etched, rinsed, dried, and rewetted with Aqua-Prep (Bisco), a non-fluoridated rewetting agent, and then bonded with One-Step. Treatment for Group III was similar to Group II, except that Aqua-Prep F (Bisco), a fluoridated rewetting agent was used. Bonded cavities were restored with a non-fluoride-containing flowable composite (AEliteFlo, Bisco). Artificial carious lesions were induced in these specimens, from which multiple 100+/-20 microm thick longitudinal sections were prepared, yielding 16 specimens per group for evaluation with polarised light microscopy (PLM) and microradiography (MRG). Representative sections were processed for transmission electron microscopy (TEM) examination and scanning transmission electron microscopy/energy dispersive X-ray (STEM/EDX) analyses. RESULTS: The differences in demineralisation of dentine among the groups were not statistically significant for 'relative' lesion depth (p > 0.05, ANOVA, Student-Neuman-Keuls test), but highly significant for 'relative' lesion area (p < 0.001). Wall lesions were consistently present in Group I, while inhibition zones were invariably observed in Group III. 87.5% of Group II specimens exhibited neither wall lesion nor inhibition zone. TEM showed that remnant dentine apatite crystallites within the inhibition zones in Group III were larger and denser than those present within the corresponding wall lesions. STEM/EDX analyses confirmed the presence of calcium, phosphorus and fluorine in these plate-like crystallites. CONCLUSION: When used with a water-free, single-bottle dentine adhesive, a non-fluoridated rewetting agent is able to reduce, but cannot completely prevent recurrent caries. The use of a fluoridated rewetting agent is useful under the situation when microleakage occurs, by providing the additional benefit of fluoride-induced demineralisation inhibition.