Arachidonic acid is metabolized to three distinct classes of metabolites: cyclooxygenase produces prostaglandins, prostacyclins, and thromboxanes; lipoxygenase produces hydroperoxyeicosatetraenoic acids and, epoxygenase, a NADPH-dependent cytochrome P-450 enzyme, produces epoxyeicosatrienoic acids. Addition of 5,6-epoxyeicosatrienoic acid (5,6-EET) to GH3 cells, a rat anterior pituitary cell line, produces a rapid, dose-dependent stimulation of prolactin (PRL) release. Incubation with arachidonic acid (AA) was ineffective at increasing PRL release. The lipoxygenase metabolite 5-hydroxyeicosatetraenoic acid (5-HETE), however, increased PRL release from GH3 cells but with a much lower maximal response than 5,6-EET. We examined the role of metabolism inhibitors in 5,6-EET-mediated PRL release. Microsomal and cytosolic epoxide hydrolase (EH) inhibitors do not alter 5,6-EET-induced PRL release, suggesting that EH does not play a significant role in 5,6-EET mediated PRL release from GH3 cells. A chemical analog of 5,6-EET wherein the epoxide oxygen is replaced with a sulfur to afford 5,6-thioepoxyeicosatrienoic acid was also tested and found to stimulate the release of PRL, although not to the same extent as 5,6-EET. Although 5-HETE tends to increase PRL release from GH3 cells, 5,6-EET is significantly more potent at the stimulation of PRL release from GH3 cells.