Exposure of plants to low, non-freezing temperatures results in genetically programmed changes in the physiology and biochemistry of plants that are critical for low-temperature survival. While genomic and transcript-profiling studies have provided a wealth of information about the process of cold acclimation, there is growing recognition that the abundance of mRNA transcripts is not always representative of cognate protein levels and that mechanisms of post-translational regulation must also play an important role. Recent advances in proteomic technologies have greatly increased the utility of studying global changes in proteins and significantly increased the efficiency and reliability of bidimensional gel electrophoresis (2DE). This has been accompanied by advances in mass spectrometry (MS) and in protein-sequence databases used in the identification of separated proteins. Although encumbered with its own constraints, proteomics has become a powerful method used to study the relationship between gene expression (transcriptomics) and metabolism (metabolomics). New techniques in gel-based approaches, such as difference gel electrophoresis (DIGE), are now available to provide both qualitative and quantitative data about the differential expression of proteins. Following 2DE separation and analysis, selected spots are usually subjected to tryptic digestion and identified using electrospray ionization, matrix-assisted laser desorption/ionization-time of flight-MS and/or tandem MS. In this review, the advantages of the DIGE technique over existing 2DE techniques will be presented, and the utility of 2DE techniques in low-temperature studies as a complement to transcriptomics, genomics and metabolomics will be discussed. Examples of proteomic studies utilizing different tissues or subcellular compartments in response to cold will also be presented.