Rhizosphere processes have a major impact on copper (Cu) availability and its fractions in soils. A greenhouse experiment with wheat was performed to investigate availability (using seven chemical procedures) and fractionation of Cu in the rhizosphere of ten agricultural soils (Typic Calcixerepts) amended with sewage sludge (1% w/w) using rhizoboxes. The results show that available Cu concentrations in rhizosphere soils were significantly (P < 1%) lower than in bulk soils. In comparison with the bulk soils, in the rhizosphere soils the concentration of Cu associated with organic matter and residual Cu increased by 24 and 4%, respectively, whereas exchangeable Cu, Cu associated with iron-manganese oxides, and Cu associated with carbonate decreased by 20, 14, and 12%, respectively. Dissolved organic carbon (DOC) and Cu associated with iron-manganese oxides and Cu associated with organic matter in the rhizosphere and bulk soils were significantly correlated (P < 5%). The results show that the differences between rhizosphere and bulk soils in chemical conditions such as DOC concentrations can change the proportion of soil Cu fractions and, therefore, Cu availability for wheat in calcareous soils amended with sewage sludge. The results show that the wheat root-induced modifications of chemical and biological soil conditions do not only lead to Cu depletion in mobile soil Cu fractions, but also to modification in soil Cu fractions which are commonly considered as more stable.