Arachidonic acid (AA) is proposed to be a facilitatory retrograde messenger in hippocampal glutamatergic synapses. In this study, we found that AA (10 μM) increased the basal outflow (19 ± 4%) and the K+-evoked release of [3H]GABA (38 ± 3%) from rat hippocampal synaptosomes. This effect is likely to be a direct action of AA, as it was not mimicked by arachidic acid (10 μ M) and was not modified by inhibition of either lipooxygenase with nordihydroguaiaretic acid (50 μM) or cyclooxygenase with indomethacin (100 μM). Activation of protein kinase C may be involved, as chelerythrine (6 μM), a protein kinase C inhibitor, attenuated the AA (10 μM)-facilitation of K+-evoked [3H]GABA release by 58 ± 5%. Phospholipase A2 (2 U/mL), an enzyme that releases AA, and melittin (1 μM), a phospholipase A2 activator, mimicked the AA-facilitation of evoked [3H]GABA release (70 ± 6% and 76 ± 7% facilitation, respectively). These results show that exogenously added and endogenously produced AA increased basal outflow and K+-evoked release of [3H]GABA from rat hippocampal synaptosomes. Thus, AA can no longer be considered solely a facilitatory neuromodulator in the hippocampus, as this AA-facilitation of the release of the main inhibitory neurotransmitter may predominate under certain circumstances.