We have previously found that glyceroneogenesis is very active in brown adipose tissue (BAT) and increases in fasted, diabetic and high-protein-diet-fed rats, situations of reduced thermogenic activity. To understand better the role of glyceroneogenesis in BAT glycerol-3-phosphate (G3P) generation, we investigated its activity during cold exposure (10 days at 4 degrees C), a condition in which, in contrast to the above situations, BAT thermogenesis is markedly activated. Rates of total (from all sources) BAT fatty acid (FA) synthesis and rates of incorporation of glucose carbon into BAT glyceride-FA and -glycerol in vivo were markedly increased by cold exposure. Cold exposure induced a marked increase in BAT glyceroneogenic activity, evidenced by (1) increased rates of non-glucose carbon incorporation into glyceride-glycerol in vivo and of [1-14C]-pyruvate incorporation into glyceride-glycerol in vitro, and (2) a threefold increase in phosphoenolpyruvate carboxykinase activity. Most of the glyceride-glycerol synthesized by BAT via glyceroneogenesis or from glucose was used to esterify preformed FA. This use was markedly increased by cold exposure, in parallel with a pronounced activation of BAT lipoprotein lipase activity. In conclusion, during cold exposure BAT glyceroneogenesis is markedly activated, contributing to increase the generation of G3P, which is mostly used to esterify preformed FA.