The recruitment process induced by acclimation of mammals to cold includes a marked alteration in the acyl composition of the phospholipids of mitochondria from brown adipose tissue: increases in 18:0, 18:2(n-6) and 20:4(n-6) and decreases in 16:0, 16:1, 18:1 and 22:6(n-3). A basic question is whether these alterations are caused by changes in the concentration of uncoupling protein-1 (UCP1) or the thermogenesis it mediates - implying that they are secondary effects - or whether they are an integrated, independent part of the recruitment process. This question was addressed here using wild-type and UCP1-ablated C57Bl/6 mice acclimated to 24 °C or 4 °C. In wild-type mice, the phospholipid fatty acyl composition of mitochondria from brown adipose tissue showed the changes in response to cold that were expected from observations in other species and strains. The changes were specific, since different changes occurred in skeletal muscle mitochondria. In UCP1-ablated mice, cold acclimation induced acyl alterations in brown adipose tissue that were qualitatively identical and quantitatively similar to those in wild-type mice. Therefore, neither the increased content of UCP1 nor mitochondrial uncoupling altered the effect of cold on acyl composition. Cold acclimation in wild-type mice had little effect on phospholipid acyl composition in muscle mitochondria, but cold-acclimation in UCP1-ablated mice caused significant alterations, probably due to sustained shivering. Thus, the alterations in brown adipose tissue phospholipid acyl composition are revealed to be an independent part of the recruitment process, and their functional significance for thermogenesis should be elucidated.