Most studies on protein metabolism require extraction and fractionation of the individual proteins or polypeptides. However, when the proteins from Eucalyptus leaves are extracted by a standard procedure, commonly utilized for the leaves of many plant species, and subsequently fractionated by sodium dodecylsulphate:polyacrylamide gel electrophoresis (SDS-PAGE), no clear polypeptide bands are visible in the gel. A method has been developed and optimized that is efficient in extracting the proteins from Eucalyptus leaves, producing an excellent resolution when the polypeptides are fractionated by SDS-PAGE. This procedure involves five consecutive steps: (i) lyophilization of the leaves followed by grinding to a fine powder in liquid nitrogen; (ii) washing with methanol:acetic acid:water (10:1:9); (iii) washing with hexane; (iv) washing with acetone; (v) solubilization of the proteins in the SDS-PAGE sample buffer containing SDS and 2-mercaptoethanol. This optimized procedure allows the detection, by SDS-PAGE or fluorography, of changes in polypeptide composition in attached or detached Eucalyptus leaves during cold acclimation or heat shock. It may also be utilized successfully in the extraction of leaf proteins from other tree species. In addition, it has been possible to induce cold acclimation in detached leaves of Eucalyptus, a methodology which will speed considerably the study of polypeptide composition in response to cold.