We used gas chromatography/mass spectrometry to measure brain 12-HETE (12-Hydroxy-5,8,10,14-eicosatetraenoic acid) formation from endogenous arachidonic acid in different species and different brain regions and in isolated brain microvessels. When blood-free brain slices were incubated for 20 minutes we found that the rabbit and cat brain incubates contained little 12-HETE when compared to rat and mouse brain incubates. Further in vitro studies of various rat brain regions showed a generally even distribution of 12-HETE. When isolated rat or rabbit microvessels were incubated and analyzed, we found 1 and 0.25 micrograms, respectively, of 12-HETE/mg of microvessel protein. Also, rabbit brain had limited or no capacity to actively metabolize tritiated 12-HETE. In summary, these studies show substantial species variation with respect to brain formation of 12-HETE and indicate that the vasculature is a potentially significant contributor to the 12-HETE found in whole brain tissue.