The shell of the iron-storage protein ferritin consists of two types of subunit (heavy, Mr = 21,000; light, Mr = 19,000). To study the structure and expression of the ferritin subunit genes, recombinant plasmids containing ferritin cDNA have been isolated. A cDNA library was constructed in the vector pBR322 from rat liver polysomal mRNA and screened by using I125-labeled antibody to rat liver ferritin. Six positive clones were identified and were shown to contain cDNA inserts ranging in length from 800 to 950 base pairs. When these cDNA clones were used for hybrid selection of rat liver mRNA and the selected mRNAs were translated in vitro, the products from each clone migrated on denaturing gels in a position similar to that of the light subunit of ferritin. No evidence of translation of the heavy subunit was obtained, indicating that the two subunits are encoded by separate mRNAs. RNA blot analysis gave a length of 1,100 nucleotides for the light-subunit mRNA. One of the cDNA inserts was fractionated into four fragments by using the restriction enzyme Sau3A. When the fragments were hybridized with Southern blots of rat spleen DNA, each fragment yielded similar patterns of hybridization to that obtained with the intact cDNA. Therefore, all regions of the cDNA sequence contain homologous sequences to similar genomic restriction fragments. This is consistent with the existence of a family of genes that encode the light subunit of rat ferritin.