This study examines the incorporation of highly unsaturated n-3 fatty acids (HUFA) into triacylglycerols (TAG) of brown adipose tissue (BAT), and their effect on the positional distribution of saturated (SFA) and of unsaturated (UFA) 16- or 18-carbon fatty acids. To this end, rats were fed a fish oil diet for up to four weeks. The stereospecific analysis of TAG was based on generation of sn-1,2- and sn-2,3-acylglycerols by Grignard degradation, followed by synthesis of phosphatidic acid and specific hydrolysis with phospholipase A2. From the end of the first week of fish oil feeding, a steady-state in the fatty acid composition of TAG in BAT was reached. HUFA concentration increased 30-fold, mainly at the expense of n-9 UFA and of SFA. The amount of SFA decreased selectively at position 3, where these fatty acids were progressively replaced by n-3 HUFA. By contrast, the amount of UFA decreased at all positions, and their positional distribution was not affected. About 60% of HUFA was incorporated at position 3. Nearly twice as much 22:6n-3 was incorporated into TAG than had been previously observed in white adipose tissue (WAT) [Leray, C., Raclot, T., and Groscolas, R. (1993) Lipids 28, 279-284]. At the steady-state, the distribution of HUFA was characterized by high proportions of 22:6n-3 and 20:5n-3 in position 3. Moreover, in each position of TAG, a steady level was reached rapidly (within 1 wk).(ABSTRACT TRUNCATED AT 250 WORDS)