Heme oxygenase oxidatively cleaves heme to biliverdin, leading to the release of iron and CO through a process in which the heme participates both as a cofactor and as a substrate. Here we report the crystal structure of the product, iron-free biliverdin, in a complex with human HO-1 at 2.19 A. Structural comparisons of the human biliverdin-HO-1 structure with its heme complex and the recently published rat HO-1 structure in a complex with the biliverdin-iron chelate [Sugishima, M., Sakamoto, H., Higashimoto, Y., Noguchi, M., and Fukuyama, K. (2003) J. Biol. Chem. 278, 32352-32358] show two major differences. First, in the absence of an Fe-His bond and solvent structure in the active site, the distal and proximal helices relax and adopt an "open" conformation which most likely encourages biliverdin release. Second, iron-free biliverdin occupies a different position and orientation relative to heme and the biliverdin-iron complex. Biliverdin adopts a more linear conformation and moves from the heme site to an internal cavity. These structural results provide insight into the rate-limiting step in HO-1 catalysis, which is product, biliverdin, release.