Singlet molecular oxygen ( 1 O 2 ) has well-established roles in photosynthetic plants, bacteria and fungi 1–3 , but not in mammals. Chemically generated 1 O 2 oxidizes the amino acid tryptophan to precursors of a key metabolite called N-formylkynurenine 4 , whereas enzymatic oxidation of tryptophan to N-formylkynurenine is catalysed by a family of dioxygenases, including indoleamine 2,3-dioxygenase 1 5 . Under inflammatory conditions, this haem-containing enzyme is expressed in arterial endothelial cells, where it contributes to the regulation of blood pressure 6 . However, whether indoleamine 2,3-dioxygenase 1 forms 1 O 2 and whether this contributes to blood pressure control have remained unknown. Here we show that arterial indoleamine 2,3-dioxygenase 1 regulates blood pressure via formation of 1 O 2 . We observed that in the presence of hydrogen peroxide, the enzyme generates 1 O 2 and that this is associated with the stereoselective oxidation of l-tryptophan to a tricyclic hydroperoxide via a previously unrecognized oxidative activation of the dioxygenase activity. The tryptophan-derived hydroperoxide acts in vivo as a signalling molecule, inducing arterial relaxation and decreasing blood pressure; this activity is dependent on Cys42 of protein kinase G1α. Our findings demonstrate a pathophysiological role for 1 O 2 in mammals through formation of an amino acid-derived hydroperoxide that regulates vascular tone and blood pressure under inflammatory conditions.