The aims of this study were to assess Cu, Zn, and Cr pollution in a highly polluted river in Argentina (Matanza-Riachuelo) and to evaluate tolerance strategies and toxic effects in aquatic macrophytes. Chemical techniques were used to assess the bioavailability of these metals and to evaluate their uptake and translocation by plants. The ultrastructure of the roots of a free-floating plant (Eichhornia crassipes) and the leaves of an emergent macrophyte (Sagittaria montevidensis) was examined using transmission electron microscopy. In the lower basin of the river, the highest concentrations of total heavy metals were detected in water (179 µgZn/g; 54 µgCu/g; 240 µgCr/g) and sediments (1499 µgZn/g; 393 µgCu/g; 4886 µgCr/g). In the upper basin of the river, low percentages of Zn and Cu (8 to 25%) were extracted with DTPA and EDTA, probably due to the lithogenic origin of these metals. Higher extraction percentages (24 to 66%) were obtained in the lower basin, in accordance with anthropogenic pollution. For Cr, extraction percentages were low in the upper basin of the river ( 4.5%) and extremely low in the lower basin ( 0.03%). In S. montevidensis, the BCF (bioconcentration factor) and TF (translocation factor) indexes were compatible with heavy metal exclusion mechanisms in sediments, whereas in the E. crassipes, root compartmentalization could be the main tolerance strategy. The leaves of S. montevidensis showed no evidence of damage, whereas ultrastructural alterations (plasmolyzed cells, disorganized membranes) were observed in E. crassipes.