The essential fatty acids do not have identical roles in nutrition. Linoleic acid (LA) accumulates throughout the body of most mammals, whereas α-linolenic acid (ALA) is rarely found in tissue lipids to the same extent as LA. It has been argued that this is the result of metabolism of ALA to docosahexaenoic acid (DHA) or that ALA is rapidly β-oxidized to acetyl CoA and CO2. In this study, we consider the effect of high and low ALA levels on the tissue distribution of ALA and other n-3 polyunsaturated fatty acids (PUFA) in all tissues. Guinea pigs were fed one of two defined diets for 3 wk from weaning with both diets containing 1.8% (by weight) of LA and either 1.7% ALA or 0.03% ALA. The high ALA diet was associated with significantly increased ALA levels in all tissues except the brain and significantly increased levels of long-chain n-3 PUFA in all tissues except intestines, brain, carcass, and skin. The long-chain n-3 PUFA content of the whole body was less than 5% of that of the ALA content in both diet groups, and the major long-chain n-3 PUFA (>66% of total) in the body was 22:5n-3. The brain was the only tissue where the DHA content exceeded that of 22:5n-3. On the low ALA diet, there appeared to be conservation of ALA based on a comparison of the ratio of LA to ALA in the tissues compared with that in the diet. On the high ALA diet there was a loss of ALA relative to LA in the tissues compared with the diet. These studies suggest that the low levels of tissue ALA in the guinea pig are likely the result of β-oxidation or excretion via the skin and fur rather than metabolism to DHA.