The vasodilatory effect of 20-hydroxyeicosatetraenoic acid (20-HETE) on lung arteries is opposite to the constrictor effect seen in cerebral and renal vessels. These observations raise questions about the cellular localization of 20-HETE-forming isoforms in pulmonary arteries and other tissues. Using in situ hybridization, we demonstrate for the first time CYP4A (a family of cytochrome P-450 enzymes catalyzing formation of 20-HETE from the substrate arachidonic acid) mRNA in pulmonary arterial endothelial and smooth muscle cells, bronchial smooth muscle and bronchial epithelial cells, type I epithelial cells, and macrophages in adult male rat lungs. Moreover, we detect CYP4A protein in rat pulmonary arteries and bronchi as well as cultured endothelial cells. Finally, we identify endogenously formed 20-HETE by using fluorescent HPLC techniques, as well as the capacity to convert arachidonic acid into 20-HETE in pulmonary arteries, bronchi, and endothelium. These data show that 20-HETE is an endogenous product of several pulmonary cell types and is localized to tissues that optimally position it to modulate physiological functions such as smooth muscle tone or electrolyte flux.