A coculture system was employed to study the interactions between endothelium and vascular smooth muscle cells in arachidonic acid metabolism. Bovine aortic endothelial cells grown on micropore filters impregnated with gelatin and coated with fibronectin are mounted on polystyrene chambers and suspended over confluent smooth muscle cultures. The endothelial basal laminae are oriented toward the underlying smooth muscle, and the two layers are separated by only 1 mm. Each cell layer was assayed individually: apical and basolateral fluid also was collected separately for assay. Fatty acids, including arachidonic acid, are readily transferred between the endothelial and smooth muscle cells in this system. Distribution of the incorporated fatty acids among the lipids of each cell is the same as when the fatty acid is added directly to the culture medium. Arachidonic acid released from endothelial cells is available as a substrate for prostaglandin production by smooth muscle. In addition, fatty acids released from the smooth muscle cells can pass through the endothelium and accumulate in the fluid bathing the endothelial apical surface. These fatty acid interchanges may be involved in cell-cell signaling within the vascular wall, the clearance of lipids from the vascular wall, or the redistribution of arachidonic acid and other polyunsaturated fatty acids between adjacent cell types. Furthermore, the findings suggest that prostaglandin production by smooth muscle cells can occur in response to stimuli that cause arachidonic acid release from endothelial cells.