Soybean [Glycine max (L.) Merr.] cell suspensions have the capacity to develop tolerance to excess copper, constituting a convenient system for studies on the mechanisms of copper tolerance. The functional cell organisation changes observed in these cell cultures after both short-term (stressed cells) and long-term (acclimated cells) exposure to 10 μm CuSO4 are reported from structural, cytochemical and microanalytical approaches. Cells grown in the presence of 10 μm CuSO4 shared some structural features with untreated cells, such as: (i) a large cytoplasmic vacuole, (ii) chloroplasts along the thin layer of cytoplasm, (iii) nucleus in a peripheral location exhibiting circular-shaped nucleolus and a decondensed chromatin pattern, and (iv) presence of Cajal bodies in the cell nuclei. In addition, cells exposed to 10 μm CuSO4 exhibited important differences compared with untreated cells: (i) chloroplasts displayed rounded shape and smaller size with denser-structured internal membranes, especially in copper-acclimated cells; (ii) no starch granules were found within chloroplasts; (iii) the cytoplasmic vacuole was larger, especially after long-term copper exposure; (iv) the levels of citrate and malate increased. Extracellular dark-coloured deposits with high copper content attached at the outer surface of the cell wall were observed only in cells exposed to a short-term copper stress. Structural cell modifications, mainly affecting chloroplasts, accompanied the short-term copper-induced response and were maintained as stable characters during the period of adaptation to excess copper. Vacuolar changes accompanied the long-term copper response. The results indicate that the first response of soybean cells to excess copper prevents its entry into the cell by immobilising it in the cell wall, and after an adaptive period, acclimation to excess copper may be mainly due to vacuolar sequestration.M Bernal is recipient of a predoctoral fellowship from Consejo Superior de Investigaciones Científicas (I3P Programme financed by the European Social Fund). This work was supported by the Aragón Government (Grant P015/2001 and GC DGA 2004 programme) and the Ministry of Education and Culture of Spain (BFU2005-07422-C02-01; AGL2005-05104; BFU2005-01094).Peer reviewed