1. We have previously shown that carbon monoxide (CO) potently relaxes the lamb ductus arteriosus and have ascribed this response to inhibition of a cytochrome P450-based mono-oxygenase reaction controlling the formation of endothelin-1 (ET-1). In the present study, we have examined whether CO is formed naturally in the vessel. 2. The CO-forming enzyme, haem oxygenase (HO), was identified in ductal tissue in its constitutive (HO-2) and inducible (HO-1) isoforms by Western immunoblotting and immunological staining procedures (both light and electron microscopy). HO-1 was localized to endothelial and muscle cells, while HO-2 was found only in muscle cells. Inside the muscle cells, HO-1 and HO-2 immunoreactivity was limited to the perinuclear region, and the Golgi apparatus in particular. However, upon exposure to endotoxin, HO-1 became more abundant, and both HO isoforms migrated towards the outer region of the cytoplasm close to the sarcolemma. 3. CO was formed enzymatically from added substrate (hemin, 50 microM) in the 10,000 g supernatant of the ductus and its formation was inhibited by zinc protoporphyrin IX (ZnPP, 200 microM). 4. ZnPP (10 microM) had no effect on the tone of the ductus under normal conditions (2.5 to 95% O2), but it contracted the endotoxin-treated ductus (at 2.5% O2). At the same concentration, ZnPP also tended to contract the hypoxic vessel (zero O2). 5. ZnPP (10 microM) curtailed the relaxant response of the oxygen (30%)/indomethacin (2.8 microM)-contracted ductus to bradykinin (35 nM), while it left the sodium nitroprusside (35 nM) relaxation unchanged. 6. We conclude that CO is formed in the ductus and may exert a relaxing influence when its synthesis is upregulated by an appropriate stimulus.