Changes in leaf soluble proteome were explored in 3-month-old plants of Cu-tolerant (metallicolous, M) and sensitive (non-metallicolous, NM) Agrostis capillaris L. populations exposed to increasing Cu concentrations (1-50 µM). Aims of the study were to investigate (1) differential protein expression in NM and M leaves under increasing Cu exposure, and (2) molecular mechanisms underlying higher Cu tolerance in M plants. Plants were cultivated on perlite (CuSO4 spiked-nutrient solution); fresh leaf aliquots were frozen in liquid nitrogen. Soluble leaf proteins were extracted by the trichloroacetic acid/acetone procedure, and separated with 2-DE (linear 4-7 pH gradient). Analysis of CCB-stained gels (PDQuest) reproducibly detected 214 spots, of which 107 were analyzed using LC-MS/MS.