In this work, were studied the conditions of erucic acid (cis-docosenoic, n-9) shortening by using Rat liver mitochondrial preparations which were incubated in vitro with [14-14C] erucic acid (22:1), with inhibitors of the respiratory chain (rotenone, cyanide) or not, with activators of either the shortening reaction (NAD+, NADP+), or beta-oxidation (malate, carnitine, cytochrome c) or not. The shortening activity was measured by the amount of 14C radioactivity recovered in the shorter fatty acids formed (20:1, 18:1, 16:1) when beta-oxidation was inhibited. The beta-oxidation activity was measured by the amount of 14C recovered in the acid-soluble products (P A S). The incubations were performed under conditions which were the least favourable to peroxysomal activity. Data showed that, with increasing amounts of albumin, which inhibits peroxysomal activity, increasing amounts of shorter fatty acids (20:1, 18:1, 16:1) were formed from erucic acid. This shortening reaction was strongly stimulated by NAD+, more than by NADP+; it was also stimulated by cytochrome c and much more when both NAD+ and cytochrome c were added. Similar data were observed in beta-oxidation, except that practically NADP+ did not exhibit any stimulating effect. Oxidation of NADH by mitochondria only occurred when cytochrome c was added to the medium and was not modified by the addition of ADP or rotenone. These data show that liver mitochondria are capable of shortening erucic acid, as are peroxysomes. This shortening reaction is highly NAD+-dependent and seems to be localized outside the matrix. This system could constitute a second route for utilization of fatty acids in mitochondria, besides the well-known path of beta-oxidation.