Young male Sprague-Dawley rats were fed diets containing added pyridoxine .cntdot. HCl at 22 mg/kg (control), 0 mg/kg or 88 mg/kg for 6 wk. In comparison with control or pyridoxine-supplemented (+ PN) rats, growth of the pyridoxine-deficient (-PN) rats was significantly less after 2 wk. After 6 wk, liver wt was higher but thymus and epididymal fat weights, the relation to body wt, were significantly lower in -PN compared to control rats. In -PN rats, phospholipid levels of linoleic and .gamma.-linolenic acids were increased, but arachidonic acid was decreased compared to controls in plasma, liver, thymus and skin. In liver triglycerides from -PN rats, all essential fatty acids (n3 and n6) were increased compared to both control and +PN rats. The n3 essential fatty acids were significantly increased in plasma, liver and thymus phospholipids in the +PN compared to control rats. These results support previous reports of an effect of pyridoxine on essential fatty acid metabolism and suggest that both linoleic desaturation and .gamma.-linolenic acid elongation may be impaired in -PN rats. The accumulation of essential fatty acids in the liver triglycerides of -PN rats suggests that essential fatty acid turnover between triglyceride and phospholipid may be influenced by pyridoxine.