Background: This study tested the dietary level of alpha-linolenic acid (alpha-LNA, 18:3n-3) required to maintain brain C-14-Docosahexaenoic acid (DHA, 22:6n-3) metabolism and concentration following graded alpha-LNA reduction. Methods: Fischer-344 (CDF) male rat pups (18-21 days old) were randomized to the AlN-93G diet containing as a % of total fatty acids, 4.6% ("n-3 adequate"), 3.6%, 2.7%, 0.9% or 0.2% ("n-3 deficient") alpha-LNA for 15 weeks. Rats were intravenously infused with C-14-DHA to steady state for 5 min, serial blood samples collected to obtain plasma, and brains excised following microwave fixation. Labeled and unlabeled DHA concentrations were measured in plasma and brain to calculate the incorporation coefficient, k*, and incorporation rate, J(in). Results: Compared to 4.6% alpha-LNA controls, k* was significantly increased in ethanolamine glycerophospholipids in the 0.2% alpha-LNA group. Circulating unesterified DHA and brain incorporation rates (J(in)) were significantly reduced at 0.2% alpha-LNA. Brain total lipid and phospholipid DHA concentrations were reduced at or below 0.9% alpha-LNA. Conclusion: Threshold changes for brain DHA metabolism and concentration were maintained at or below 0.9% dietary alpha-LNA, suggesting the presence of homeostatic mechanisms to maintain brain DHA metabolism when dietary alpha-LNA intake is low. (C) 2015 Elsevier Ltd. All rights reserved.