Purpose: This laboratory study investigated the effects of PVP-iodine solutions with varying pH and calcium concentrations on enamel remineralization and fluoridation by subsequent treatment with fluoride varnish. Materials and Methods: Caries-like lesions were created in bovine enamel specimens (n = 15 per group) and characterized using Vickers surface microhardness (VHN). Specimens were treated with 10% PVP-iodine solutions varying in calcium concentration (0/10/100 mM) and pH (3.0/4.0/5.0), followed by 5% sodium fluoride varnish. A fluoride varnish-only control group was included. Specimens were then placed into artificial saliva for 16 h. The varnish was removed, hardness measured and enamel fluoride uptake (EFU) determined using the microbiopsy technique. Data were analyzed with one-way ANOVA. Results: Groups receiving PVP-iodine pre-treatments exhibited directionally greater rehardening (range: p = 0.0001 - 0.7008) and EFU (p = 0.0001-0.2670) than the control group. The presence of calcium in the pre-treatment enhanced rehardening. the groups '10mM Ca/pH 3.0' (∆VHN = 10.5 ± 6.3), '100mM Ca/pH 3.0' (∆VHN = 9.7 ± 4.1) and '10mM Ca/pH 5.0' (∆VHN = 8.7 ± 7.0) displayed the highest numerical gain vs the control (∆VHN=3.6±2.2). Different pH values had a minor effect on rehardening and EFU. The calcium effect was more pronounced for EFU than for rehardening with all three '100 mM Ca' groups exhibiting higher EFU (7.0 - 7.2 µg F/cm2) than all other groups (6.1 - 6.9 µg F/cm2). Conclusion: PVP-iodine pre-treatments can be modified to enhance the rehardening and fluoridating effect of fluoride varnishes, thereby potentially improving their ability to prevent caries in vivo. Although numerical differences between groups were small, the addition of high concentrations of calcium paired with a low pH appears most favorable under the present conditions. Keywords: calcium, dental caries, fluoride varnish, PVP-iodine, remineralization