Many plants, including Arabidopsis, increase their freezing tolerance in response to low, non-freezing temperatures. This process is known as cold acclimation and involves many complex biochemical changes at the level of the metabolome. Our goal was to examine the effects of cold acclimation on the metabolome using a non-targeted metabolic fingerprinting approach. Multivariate data analyses indicate that, in Arabidopsis, a global reprogramming of metabolism occurs as a result of cold acclimation. By measuring an entire spectrum of putative metabolites based on mass-to-charge (m/z) ratios, vs. an individual or group of metabolite(s), a comprehensive, unbiased assessment of metabolic processes relative to cold acclimation was determined. Whereas leaves shifted to low temperature present metabolic profiles that are constantly changing, leaves developed at low temperature demonstrate a stable complement of components. Although it appears that some metabolic networks are modulated by the environment, others require development under low-temperature conditions for adjustment. Understanding how metabolism as a whole is regulated allows the integration of cellular, physiological and ecological attributes in a biological system, a necessity if complex traits, such as freezing tolerance, are to be modified by breeding or genetic manipulation.