This study extends our previous work (Raclot, T., and R. Groscolas. 1993. J. Lipid Res. 34: 1515-1526) which demonstrated that in the fed state fatty acids are selectively released from white adipocytes in vitro. It aims at determining whether such selectivity operates in vivo during energy depletion and has physiological relevance. This question was examined in rats by simultaneously measuring, after 1, 7, or 10 days of fasting, the fatty acid content of retroperitoneal adipose tissue (RP), and the composition of fatty acids released by isolated RP adipocytes. A preliminary dietary manipulation (fish oil feeding) allowed us to study the mobilization of a wide spectrum of fatty acids. Fasting resulted in a relative depletion of adipose tissue in fatty acids such as alpha-linolenic, arachidonic, and eicosapentaenoic, and in a relative enrichment in all very long chain saturated and monounsaturated fatty acids. After a 56% depletion of total fatty acids, 20% (22:1n-11) to 90% (20:5n-3) of the initial mass of individual fatty acids was lost. The in vivo relative mobilization of fatty acids (% in lost fatty acids / % in RP triacylglycerols) ranged from 0.31 to 2.54. For a given chain length it increased with unsaturation whereas for a given degree of unsaturation it decreased with chain length. The in vitro relative mobilization of fatty acids (% in released fatty acids/% in RP triacylglycerols) was similarly dependent on their molecular structure and, to a significant extent, directly related to in vivo mobilization. It is concluded that during fasting-induced energy depletion, the net in vivo mobilization of fatty acids from adipose tissue is selective. The selectivity of mobilization i) is based on the molecular structure of fatty acids, ii) is fully accounted for by their selective release from adipocytes, iii) leads to a profound remodelling of the composition of adipose tissue fatty acids, and iv) does not seem directed towards a preferential retention or sparing of particular fatty acids.