Zinc isotopic fractionation during adsorption onto a purified humic acid (PHA), an analogue of organic matter (OM), has been investigated experimentally as a function of pH. The Donnan Membrane device (DM) was used to separate Zn bound to the PHA from free Zn2+ ions in solution and allowed us to measure the isotopic ratios of free Zn2+. Below pH 6, adsorption of Zn on the PHA resulted in no measurable isotopic fractionation, while at higher pH, Zn bound to PHA was heavier than free Zn2+ (delta66Zn(PHA-FreeZn2+) = +0.24 per thousand +/- 0.06 2sigma, n = 4). This can be explained by changes in Zn speciation with pH, with higher complexation constants and shorter bond lengths for Zn-PHA complex compared to the free Zn2+. Complexation of Zn with PHA occurred mostly through binding to high affinity sites (HAS) and low affinity sites (LAS). Fractionation factors for 66Zn/64Zn ratios determined by mass balance calculations were equal to 1.0004 for HAS (alpha(HAS-solution)) and 1.000 for LAS (alpha(LAS-soultion)). delta66Zn(OM-FreeZn2+) should then vary according to the heterogeneous nature of the OM, because of the variable relative proportions of these two types of sites. The NICA-Donnan model, along with these fractionation factors and measured delta66Zn(TotalDissolved), was used to simulate the corresponding isotopic composition of free Zn2+ in the Seine River, France: delta66Zn(TotalDissolved-FreeZn2+) varied from +0.02 per thousand to +0.18 per thousand, depending on the HAS/LAS ratio assumed for the OM. This study allows a better understanding of Zn isotope fractionation mechanisms associated with organic mater binding.