Povidone-iodine solutions prepared to various concentrations (0.01, 0.1, 1 and 10%) with 0.2M phosphate buffer (pH 7.0) (PVP-I PB) were analyzed to determine their free iodine concentrations using membrane permeation cells, and their inactivation effects on three viruses (influenza A virus, poliovirus type 1 and adenovirus type 3) were examined. The free iodine concentrations in the 0.01-10% PVP-I PB were determined to be 1.84, 4.88, 1.58 and 0.17 ppm (approximate values), respectively, with the maximum obtained for the 0.1% solution. The virucidal efficacy of these PVP-I PB against poliovirus type 1 and adenovirus type 3 was found to be generally dependent on free iodine concentration, with the 0.1% solution being the most effective. Influenza A virus was inactivated with an action time of 15 s at all four concentrations examined. The results of this study suggested an association between free iodine concentration and virucidal efficacy for the 0.01-10% PVP-I PB.