Although nonsymbiotic hemoglobins (Hbs) are found in different tissues of dicots and monocots, very little is known about hb genes in monocots and the function of Hbs in nonsymbiotic tissues. We report the cloning and analysis of two rice (Oryza sativa L.) hb genes, hb1 and hb2, that code for plant Hbs. Rice hb1 and hb2 genes contain four exons and three introns, as with all of the known plant hb genes. At least three copies of the hb gene were detected in rice DNA, and analysis of gene expression shows that hb1 and hb2 are expressed in leaves but only hb1 is expressed in roots. A cDNA for rice Hb1 was expressed in Escherichia coli, and the recombinant Hb (rHb1) shows an unusually high affinity for O2 because of a very low dissociation constant. The absorbance spectra of the ferric and deoxyferrous rHb1 indicate that, in contrast to symbiotic Hbs, a distal ligand is coordinated to the ligand-binding site. Mutation of the distal His demonstrates that this residue coordinates the heme Fe of ferric and deoxyferrous rHb1 and stabilizes O2 in oxy-rHb1. The biochemical properties of rice rHb1 suggest that this protein probably does not function to facilitate the diffusion of O2.