Heme oxygenase, an essential enzyme of heme catabolism, is inducible by its substrate heme, by heavy metals, and by various other substances. To study the molecular mechanisms of the induction of heme oxygenase, we isolated the heme oxygenase gene from a rat genomic DNA library using cloned cDNA as hybridization probes and determined its complete nucleotide sequence. The gene is composed of 6830 nucleotides, and is organized in four introns and five exons. The transcription initiation site was identified by S1 nuclease mapping analysis. Using HeLa cell lysate, we confirmed that the transcription of cloned heme oxygenase gene is initiated accurately at the assigned initiation site. In the 5'-flanking region of the heme oxygenase gene, we found several potential binding sites for different transcription factors: a transcription factor Sp1, a positive regulator for the control of amino acid synthesis (GCN4), a heat shock transcription factor, and a metal-dependent transcription factor. Furthermore, the intron 1 contains the sequence that shows about 65% homology to that of the neuronal identifier sequence, a possible enhancer element.