Heme Oxygenase 1 (HO-1) is well known for its function as the rate limiting step in heme degradation, and is induced by a variety of stimuli such as heavy metals, inflammatory mediators, and oxidants. Recently HO-1 has been identified to have antioxidant properties and its induction is thought to be a protective mechanism. We and others have identified HO-1 as a hyperoxia inducible protein, and in human pulmonary cells this induction is dramatically enhanced by increasing cellular free iron. In this study, we further characterized the response of human HO-1 gene expression in isolated human pulmonary artery endothelial cells (HPAEC) to hyperoxia, FeSO4, and the free iron chelator, desferoximine (DFO). Total RNA was isolated following treatments with the above and HO-1 mRNA levels evaluated by Northern analysis utilizing a radiolabeled human HO-1 cDNA. The 3 fold induction by hyperoxia and potentiated by iron (15 fold induction) was completely inhibited by DFO. In addition, DFO exposure resulted in HO-1 mRNA levels below basal expression. To determine if DFO's inhibitory effect was on the HO-1 promoter, transient transfection experiments were performed using a construct containing 4.5 kb of the human HO-1 promoter in a promoterless human growth hormone (hGH) plasmid. DFO exposure resulted in a decrease in the reporter gene expression, hGH mRNA and levels, as compared to control cells. To determine if hyperoxia induction was transcriptionally dependent, nuclear run-on experiments were performed on HPAEC exposed to room air or hyperoxia plus iron. We observed a 7.3 fold increase in newly synthesized HO-1 transcripts in response to hyperoxia plus iron. Studies using the -4.5 kb human HO-1/hGH plasmid showed no promoter activity for hyperoxia with or without iron. However, there was an increase in the reporter gene expression in response to heme or cadmium indicating that this was a functional promoter. These studies indicate that free cellular iron, chelatable iron, is a mediator of HO-1 expression, and this response is transcriptionally dependent. Previous studies of the mouse HO-1 gene have identified the same regulatory region for heme, cadmium, and hyperoxia stimulation. The response of the human HO-1 gene to hyperoxia is also transcriptionally dependent, however, unlike the mouse gene, hyperoxia dependent regulation of the human HO-1 gene is unique from heme and cadmiun. Therefore the molecular mechanisms involved in hyperoxia dependent induction for the human HO-1 gene can not be extrapolated by studies of the mouse gene.