The oxidation of [1-14C]palmitate, [1-14C]linoleate, and [1-14C]arachidonate as well as their incorporation into lipids was investigated in vitamin B6 deficient rats using liver slices and isolated mitochondria. In experiments on fatty acid oxidation in liver slices, we observed a decrease in the production of 14CO2 from radioactive palmitate and linoleate, but not from arachidonate. In the case of mitochondria, we detected no difference between the deficient rats and the controls with linoleats; however, there was an increase in the oxidation of palmitate in the deficient rats as compared to the controls. Experiments on the incorporation of radioactivity into lipids showed an increase in radioactivity from [1-14C]palmitate and [1-14C]linoleate in the triglyceride, phospholipid and cholesterol fractions of deficient rats. In the case of [1-14C]arachidonate, no difference was observed between the two groups of rats. These experiments showed that major changes occurred in metabolism of palmitate and linoleate and strongly suggested that the decrease in the arachidonate content previously observed in rat tissues could not be explained by alteration in the metabolism of this fatty acid.