The main objective of this study was to investigate the efficiency of different substrates to reduce the extraction of heavy metals concentration in a heavily contaminated soil. Two contaminated soils by Cu and Zn were used to evaluate the effectiveness of eight substrates (calcium carbonate, bentonite, kaolinite, charcoal, manganese oxide, iron oxide, zeolite, phosphate) to reduce metal availability and to study the change of metals speciation in different forms using sequential extraction technique (single step). Sequential extraction technique (single step, 0.11M acetic acid, HONH3Cl, H2O2+NH4OAc, Aqua regia) was applied on contaminated soils after and before treatment to evaluate metals speciation. Results indicate that the most effective treatments in decreasing available metal concentrations were calcium carbonate, zeolite and manganese or iron oxide. Metal sequential fractionations indicate that the exchangeable fraction of Cu and Zn in contaminated soils can be transformed into unavailable forms after chemical remediation.