The in vitro metabolism of 12(R)-hydroxyeicosatetraenoic acid was studied using freshly isolated rat liver microsomes. Ten metabolites were isolated and identified by a combination of ultraviolet spectroscopy and gas chromatography/mass spectrometry. The two major metabolites were dihydroxyeicosatetraenoic acids generated by omega/omega-1 hydroxylation. Oxidation at C-5 resulted in the formation of four leukotriene-like compounds, two of which differed from leukotriene B4 in double-bond geometry alone. The other two differed from leukotriene B4 in olefin geometry and C-5 configuration. Epoxidation at the 14,15-olefin resulted in the formation of two diastereomeric epoxy alcohols, while C-16 hydroxylation gave two diastereomeric dihydroxyeicosatetraenoic acids.