The movement of alpha-linolenic acid (C18:3, n-3) through the mitochondrial outer membrane to oxidation sites was studied in rat liver and compared with the movement of linoleic acid (C18:2, n-6) and oleic acid (C18:1, n-9). All differ in the degree of unsaturation, but have the same chain length and the same position of the first double bond when counted from the carboxyl end. The following results were obtained. (1) The overall beta-oxidation in total mitochondria was in the order C18:3, n-3 greater than C18:2, n-6 greater than C18:1, n-9, independent of the amount of albumin in the medium. (2) The rate of formation of acylcarnitine from acyl-CoA was higher with oleoyl-CoA than with linoleoyl-CoA, and remained very low with alpha-linolenoyl-CoA for all concentrations studied. (3) When the formation of acylcarnitines originated from fatty acids (as potassium salts) in a medium containing CoA and ATP, the conversion of alpha-linolenate was greater than that of linoleate, which in turn was greater than that of oleate. (4) Use of a more purified mitochondrial fraction, practically devoid of peroxisomes, did not modify the results obtained with alpha-linolenate. (5) alpha-Linolenoyl-CoA did not inhibit oxidation of labelled alpha-linolenate, whereas the other acyl-CoAs did. (6) Transfer to carnitine of all three fatty acids (as potassium salts) by carnitine palmitoyltransferase-I (CPT-I) was similarly inhibited by increasing concentrations of malonyl-CoA. (7) On using a fraction containing mitochondrial outer membranes, the formation of acylcarnitines from potassium salts of fatty acids was qualitatively and quantitatively similar to that found with whole mitochondria. (8) Our observations show that alpha-linolenoyl-CoA synthesized other than in the mitochondria cannot be used to any great extent by the mitochondria due to its configuration. However when added as the unactivated form, alpha-linolenate appears to be very quickly oxidized, but should first be activated by acyl-CoA synthetase in the mitochondrion itself. Then it is rapidly channelled to CPT-I. These enzymic sites are probably close together in the mitochondrial outer membrane. The different behaviour of the alpha-linolenic group compared with the other acyl groups in the studied pathway can be explained by a different spatial arrangement due to the number and position of the double bonds.