The sequential appearance of changes in interscapular brown adipose tissue composition, cytochrome oxidase activity and GDP binding was studied throughout pregnancy and lactation in the rat. Brown adipose tissue was hypertrophied during pregnancy because of progressive lipid accumulation, whereas its mitochondrial component and GDP binding to brown fat mitochondria were unchanged. In early lactation (day 5) there was a decrease in the overall GDP binding to brown fat only because of the lower mitochondrial protein content. In late stages of lactation (days 10 and 15), the amount of tissue and its mitochondrial protein content were minimal and the GDP binding per mitochondrial protein decreased substantially. Scatchard analysis in day-15-lactating rats indicated a large decrease in GDP binding sites without any changes in affinity. It is concluded that the diminished thermogenic activity of brown fat in lactation is attained through changes at different structural levels of the tissue occurring in a characteristic sequential trend; first a reduction in its mitochondrial component, and only later, at mid-lactation, a decrease in the specific mitochondrial proton conductance pathway activity.