The organization of two closely clustered genes, Fer1HCH and Fer2LCH, encoding the heavy-chain homolog (HCH) and the light-chain homolog (LCH) subunits of Drosophila melanogaster ferritin are reported here. The 5019-bp sequence of the cluster was assembled from genomic fragments obtained by polymerase chain reaction (PCR) amplification of genomic DNA and from sequences obtained from the Berkeley Drosophila Genome Project (BDGP) (http://www.fruitfly.org). These genes, located at position 99F1, have different exon-intron structures (Fer1HCH has three introns and Fer2LCH has two introns) and are divergently transcribed. Computer analysis of the possibly shared promoter regions revealed the presence of putative metal regulatory elements (MREs), a finding consistent with the upregulation of these genes by iron, and putative NF-kappaB-like binding sites. The structure of two other invertebrate ferritin genes, from the nematode Caenorhabditis elegans (located on chromosomes I and V), was also analyzed. Both nematode genes have two introns, lack iron-responsive elements (IREs), and encode ferritin subunits similar to vertebrate H chains. These findings, along with comparisons of ferritin genes from invertebrates, vertebrates, and plants, suggest that the specialization of ferritin H and L type chains, the complex exon-intron organization of plant and vertebrate genes, and the use of the IRE/iron regulatory protein (IRP) mechanism for regulation of ferritin synthesis are recent evolutionary acquisitions.