Heavy metals environment contamination has becoming a large worldwide problem, which affects crop productivity, soil fertility and contributes for bioaccumulation of these metals in trophic chain. Water hyacinth (Eichhornia crassipes) is an aquatic plant abundant mainly in tropical and sub-tropical regions endowed with capacity to absorb and tolerate high concentration of heavy metals. The aim of this study was to evaluate the potential of plasmalemma and tonoplast H⁺ pumps as biochemical markers for the presence of heavy metals and other river pollutants. Roots and shoots were excised from E. crassipes plants sampled from different regions of the Paraíba do Sul river (PSR) and cell fractionation was carried to obtain the plasmalemma and tonoplast vesicles. The hydrolytic activities of the H⁺ pumps in E. crassipes sampled from three regions of the river (low, medium and high RPS), in average, were two times higher than that observed in plants from a region free of pollutants (control plants from Imbé river). Although plants sampled from the different sites of PSR exhibited alterations in their initial (F₀) and maximal (Fm) fluorescence, the photochemical efficiency evaluated as the Fv/Fm ratio was not altered in relation to the control plants. Thus, this plant species can drive a non-photochemical energy dissipation in order to maintain the efficiency of its photosynthetic apparatus under stress conditions. The activation of the plasmalemma and tonoplast H⁺-ATPases as well as of the H⁺-PPase can energize the heavy metals cell compartmentation and/or exclusion processes and can be used as biochemical marker towards the use of this aquatic plants as monitors of heavy metal water pollution.