This study was designed to establish whether fluidity and lipid composition of brain membranes were affected by hypothyroidism, in the hope of clarifying the mechanism through which thyroid hormone deficiency might influence nerve cell functions. Rats were made hypothyroid by injections of PTU (dissolved in 0.005 M NaOH, 50 mg/kg, ip, daily for 28 days). Membrane fluidity, cholesterol (chol) and phospholipids (PL) content, and PL and their fatty acid composition were measured in plasma, erythrocyte plasma membranes, liver microsomes and brain subcellular fractions. P2 pellets from brains of hypothyroid animals were less fluid than those of euthyroid ones; subcellular fractionation showed that mitochondrial membranes were responsible for the rigidity observed. Similar changes were found in erythrocyte "ghosts". The reduced fluidity seemed to be related more to alterations in the ratios between phosphatidylcholine, sphingomyelin and phosphatidylethanolamine, than to those of chol/PL, protein/PL or fatty acid unsaturation index. The well known alteration in hypothyroidism-induced desaturase activity, which in peripheral tissue leads to a reduction of 20:4n-6 and to an increase in its precursors (18:2n-6 and 20:3n-6), is barely detectable in brain membranes. Only in phosphatidylcholine of synaptosomes and myelin did slight changes in percentages of the n-6 family fatty acids result in a significant alteration of 20:4n-6/20:3n-6 ratios.