Ferritin stores iron within a protein shell consisting of 24 subunits of two types, heavy (H) and light (L). According to Southern blotting, the rat genome contains four copies homologous to the H-subunit cDNA (H cDNA). To determine whether only one of these is expressed, H cDNAs isolated from rat liver and heart mRNAs were compared and found to share identical nucleotide sequences. Next, genomic clones for three of the four rat H-subunit loci were isolated. Two were classical processed pseudogenes, whereas the third contained an expressed gene. RNase intron mapping of this expressed gene generated the same exon protection pattern when total RNA from rat liver or heart was used, indicating that this gene accounts for most or all of the H-subunit mRNAs (H mRNAs) in these tissues. Comparison of the expressed rat H-subunit gene (H gene) structure with published sequences for other species displays considerable conservation. The coding sequence of the rat H gene predicts 95% similarity to the human amino acid sequence, thus being more highly conserved than the L-subunit sequence of these species. Near the cap region of the 5' untranslated region, the rat H mRNA displays a 28-nucleotide sequence that is almost totally conserved in the corresponding region of the human, bullfrog, and chicken H mRNA and is also faithfully represented in the rat and human L-subunit mRNAs (L mRNAs), thus making this sequence a prime candidate for involvement in the known translational regulation of both subunits by iron. In the 5' flanking region, partially conserved sequences common to H gene and L-subunit gene (L gene) of the rat may be involved in transcriptional regulation by iron, whereas those conserved only in the H gene of man and the rat imply that other factors may independently control H-subunit regulation.