Polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA) and arachidonic acid (AA), are the main components of the phospholipids, in cerebral membranes. A dietary-induced cerebral DHA deficit results in altered behaviour and neurotransmission in rodents. To determine whether PUFA were acting on the neurotransmitter release machinery, we measured the release of [(3)H]-noradrenaline (NA) from SH-SY5Y neuroblastoma cells with modified PUFA membrane contents and from cells incubated with medium containing high DHA or AA. The membranes of cells incubated with 70 microM DHA for 3 days had 7.6-times more DHA in their ethanolamine glycerophospholipids, while the membranes of cells incubated with AA had 40% less. Incorporation of DHA enhanced basal [(3)H]-NA release (25%, p<0.05), but not KCl-evoked [(3)H]-NA release. Brief incubation with DHA during vesicle mobilization also strongly increased [3H]-NA release. AA had no effect. The genes encoding for the calcium sensor synaptotagmin 1, and for the two SNARE complex proteins syntaxin 1A and synaptobrevin 1 were not affected by PUFA incorporation, as indicated by assays for specific mRNAs and proteins. Thus both a high membrane DHA content and free DHA in the medium enhance the release of [(3)H]-NA from SH-SY5Y cells. This suggests that brain membrane DHA influences exocytosis, which then regulates neurotransmission.