Investigation of endothelial regulation of vascular reactivity and tone has led to the discovery of chemical mediators such as nitric oxide (NO) and prostacyclin (PGI₂). Evidence has emerged indicating another as yet unidentified hyperpolarizing agent (endothelium-derived hyperpolarizing factor or EDHF) that is different from NO and PGI₂ and exerts it effects through calcium-activated potassium channels (K_Ca). Previous studies to identify EDHF have been carried out using inhibitors that block NOS and COX before application of K_Ca channel and/or muscarinic receptor antagonists. Such pharmacological manipulation has complicated interpretation of results, clearly pointing to the need for altered approaches to verify previous studies. Evidence has emerged that potential EDHF candidates vary with vessel size, species and tissue beds, indicating that there may be more than one EDHF. To date, the most commonly described and best characterized of them all are a set of arachidonic acid metabolites, epoxyeicosatrienoic acids (EETs). These compounds are synthesized both intra- and extravascularly. Until recently, methodology to detect EETs in the microvasculature has been tedious and expensive, limiting the experimentation that is necessary to confirm EETs as an EDHF. This review describes state-of-the-art methods for assaying EETs in biological samples, after summarizing evidence for EETs as an EDHF and introducing emerging concepts of the role of extravascular EETs in linking neuronal activity to localized blood flow during functional hyperemia.