The manganese-containing superoxide dismutase (MnSOD) constitutes one of the major cellular defense mechanisms against the toxic effects of superoxide radical. The development of tolerance in adult rats to lethal exposure of O2 (100%) after pre-exposing them to a sublethal concentration of O2 (85%) was found to be closely associated with the increased activity of this enzyme in the lungs. Further experiments have shown that the transcriptional rate of the gene coding for MnSOD in rat lungs is increased at day 3 of 85% O2 exposure. To elucidate the nature of this transcriptional activation during hyperoxic insults, we chose to first understand the structure of the rat MnSOD gene. Three overlapping rat genomic fragments were isolated, and the DNA sequence containing the whole MnSOD gene was completely determined. The rat MnSOD gene contains at least five exons and is located in one piece of 16.4-kb EcoRI genomic DNA fragment. However, Southern blot analysis of total rat genomic DNA probed with MnSOD cDNA revealed an additional hybridizing 8.6-kb EcoRI genomic fragment besides the 16.4-kb one. To clarify the origin of this unexpected hybridizing genomic fragment, three unique genomic sequences derived from the promoter, intron 2, and the 3' untranslated region of the genomic clones were used to rehybridize the same Southern blot filter and were found to only hybridize to the 16.4-kb but not 8.5-kb EcoRI genomic fragment. These data suggest: (1) two MnSOD genes are present per haploid rat genome, and (2) all three cloned genomic fragments are derived from the MnSOD gene, which is located in the 16.4-kb EcoRI genomic fragment.(ABSTRACT TRUNCATED AT 250 WORDS)