Heme oxygenase-1 (HO-1) induction attenuates the development of angiotensin II (Ang II)-dependent hypertension. However, the mechanism by which HO-1 lowers blood pressure is unknown. The goal of this study was to test the hypothesis that induction of HO-1 reduces Ang II-mediated increase in superoxide production in cultured thick ascending loop of Henle (TALH) cells. Studies were performed on an immortalized cell line of mouse TALH cells. HO-1 was induced in cultured mTALH cells by treatment with cobalt protoporphyrin (CoPP, 10 µM), hemin (50 µM) or by transfection with a plasmid containing the human HO-1 isoform. Treatment of mTALH cells with 10^-9 M Ang II increased dihydroethidium (DHE) fluorescence (an index of superoxide levels) from 35.5 ±5 to 136 ±18 RFU/µm². Induction of HO-1 via CoPP, hemin, or overexpression of the human HO-1 isoform significantly reduced Ang II induced DHE fluorescence to 64 ± 5, 64 ± 8, and 41 ± 4 RFU/µm², respectively. In order to determine which metabolite of HO-1 is responsible for reducing Ang II mediated increases in superoxide production in mTALH cells, cells were preincubated with bilirubin or the carbon monoxide (CO) releasing molecule, CORM-A1 (100 µMeach) prior to exposure to Ang II. DHE fluorescence averaged 80 ± 7 RFU/µm² after incubation with Ang II and was significantly decreased to 55 ± 7 and 53 ± 4 RFU/µm² after pretreatment with bilirubin and CORM-A1. These results demonstrate that induction of HO-1 in mTALH cells reduces Ang II-mediated superoxide production through both the production of bilirubin and CO.