Although it is agreed that n-3 polyunsaturated fatty acids (PUFAs) are important for brain function, it has yet to be demonstrated how they are involved in precise cellular mechanisms. We investigated the role of enhanced n-3 PUFA in astrocyte membranes on the gap junction capacity of these cells. Astrocytes isolated from newborn rat cortices were grown in medium supplemented with docosahexaenoic acid (DHA), the main n-3 PUFA in cell membranes, or arachidonic acid (AA), the main n-6 PUFA, plus an antioxidant (alpha-tocopherol or N-acetyl-cystein) to prevent peroxidation. The resulting three populations of astrocytes differed markedly in their n-3:n-6 PUFA ratios in phosphatidylethanolamine and phosphatidylcholine, the main phospholipids in membranes. DHA-supplemented cells had a physiological high n-3:n-6 ratio (1.58), unsupplemented cells had a low n-3:n-6 ratio (0.66) and AA-supplemented cells had a very low n-3:n-6 ratio (0.36), with excess n-6 PUFA. DHA-supplemented astrocytes had a greater gap junction capacity than unsupplemented cells or AA-supplemented cells. The enhanced gap junction coupling of DHA-enriched cells was associated with a more functional distribution of connexin 43 at cell interfaces (shown by immunocytochemistry) and more of the main phosphorylated isoform of connexin 43. These findings suggest that the high n-3:n-6 PUFA ratio that occurs naturally in astrocyte membranes is needed for optimal gap junction coupling in these cells.