In Saccharomyces cerevisiae, HEM13 encodes the enzyme coproporphyrinogen III oxidase, which catalyzes the rate-limiting step in heme biosynthesis. HEM13 is a regulated hypoxic gene repressed by Rox1p and Mot3p under aerobic conditions. In this study, we further investigate the hypoxic expression of HEM13, focusing on the promoter regions that are functionally important during hypoxia and on the effect of deleting the transcriptional regulators Sut1p, Sut2p, Upc2p, Ecm22p and Ixr1p. Ixr1p is necessary for the high expression of HEM13 under hypoxic conditions and its function is exerted in vivo through the HEM13 promoter region extending from -577 to -419. Ixr1p binds in vivo to the HEM13 promoter both under aerobic and under hypoxic conditions. Purified Ixr1p binds in vitro to two sequences extending from -534 to -509 and from -497 to -450, respectively. These DNA regions compete for Ixr1p binding and the consensus KTTSAAYKGTTYASA is important for the regulatory protein to interact. These results suggest that the regulation of HEM13 expression is dependent on two proteins with high mobility group (HMG) domains: Rox1p and Ixr1p. Their interactions with the HEM13 promoter might change in the transition from aerobiosis to hypoxia.