Tight junctions (TJs) are cell-to-cell contacts made of strands, which appear as ridges on P faces and complementary furrows on E faces on freeze fracture replicas. Evidences and opinions on whether these strands are composed of either membrane-bound proteins or lipid micelles are somewhat varied. In the present work we alter the lipid composition of Madin-Darby canine kidney monolayers using a novel approach, while studying (i) their transepithelial electrical resistance, a parameter that depends on the degree of sealing of the TJs; (ii) the apical-to-basolateral flux of 4 kD fluorescent dextran (JDEX), that reflects the permeability of the intercellular spaces; (iii) the ability of TJs to restrict apical-to-basolateral diffusion of membrane lipids; and (iv) the pattern of distribution of endogenous and transfected occludin, the sole membrane protein presently known to form part of the TJs. We show that changing the total composition of phospholipids, sphingolipids, cholesterol and the content of fatty acids, does not alter TER nor the structure of the strands. Interestingly, enrichment with linoleic acid increases the JDEX by 631%. The fact that this increase is not reflected in a decrease of TER, suggests that junctional strands do not act as simple resistive elements but may contain mobile translocating mechanisms.