Cardiolipins from liver, heart and kidney mitochondria of rats fed a fat-free diet for 66 days have been analyzed for their fatty acid composition and positional distribution. The main effect was a dramatic decrease of linoleic acid which was counterbalanced by increases in the levels of palmitoleic, oleic and cis-vaccenic acids. Linoleic acid remains asymmetrically distributed between positions 1(1") and 2(2") with a positive selectivity for positions 1(1"). Its decrease is considerably faster in positions 2(2") than in positions 1(1"), which would suggest different rates of fatty acid turnover. Fat deficiency induces the appearance of 18:2(n-7) and a significant increase of 20:3(n-6) (dihomo-gamma-linolenic acid) in liver and kidney cardiolipins. In contrast, 20:3(n-6) level remains unchanged in other mitochondrial phospholipids. 18:2(n-7) and 20:3(n-6) are almost evenly distributed between both pairs of positions. Both acids have a common structural feature, that is double bonds in positions 8 and 11. 20:3(n-9) accumulates in large amounts in other mitochondrial phospholipids, but not in cardiolipins. Although surprising, 20:3(n-6) has thus to be considered as a specific marker of deficiency for cardiolipins when it is esterified to positions 1(1"). Taking into account various analytical data, it would appear that positions 1(1") of cardiolipins can only incorporate unsaturated fatty acids containing at least one cis double bond in position 8 or 9, with no other double bond between these positions and the carboxylic group.