1. Whole-cell recordings from cultured rat hippocampal neurons, from freshly dissociated dorsal root ganglion (DRG) neurons and from human embryonic kidney (HEK) 293 cells expressing the glutamate receptor GluR6 subunit were used to study the modulation of kainate receptor channels by long chain fatty acids. 2. In all three cell types, application of cis-unsaturated fatty acids caused a dose-dependent reduction in whole-cell currents evoked by kainate. Docosahexaenoic acid (DHA), arachidonic acid (AA), linolenic acid and linoleic acid all produced substantial inhibition at a concentration of 50 μM, whereas inhibition by linolenelaidic acid and linolelaidic acid was significantly weaker. Fully saturated fatty acids were essentially inactive. With continuous exposure to active fatty acids, the peak current elicited by kainate declined over a time course of several minutes to reach a steady-state level less than 50% of the initial amplitude. Recovery was slow in control solution, but was speeded up by exposure to bovine serum albumin (0.5 mg ml-1), a protein that binds fatty acids with submicromolar affinity. The inhibition in neurons was half-maximal with 5-15 μM AA or DHA, but potency was at least 10-fold greater at GluR6 in HEK 293 cells. 4. Inhibition by AA or DHA was unaffected by extracellular nordihydroguaiaretic acid (10 μM), indomethacin (10 μM), 17-octadecynoic acid (30 μM) or 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine dihydrochloride (H-7; 10 μM). Furthermore, inclusion of H-7 (100 μM), BAPTA (10 mM), AA (50 μM), antioxidants, or the protein kinase C inhibitor PKC19-36 (20 μM) in the internal solution had little effect on whole-cell currents and did not prevent inhibition of currents by extracellular application of AA or DHA. 5. We conclude that the inhibition produced by cis-unsaturated fatty acids does not require conversion to oxidized metabolites or activation of PKC. Instead, active compounds may interact directly with an extracellular, or intramembraneous, site on kainate receptors.