Aging alters a variety of physiological functions of the heart. The molecular basis of the age-related functional changes has not been fully understood. Differential gene expression provides the basis for many fundamental cellular processes associated with development and aging. The identification and cloning of genes whose expression is modulated by aging can be of importance for our better understanding of these age-related phenomena. In order to isolate and characterize gene products differentially expressed in senescent hearts, we applied a differential display method for screening those genes in rat ventricular myocardium. Total RNAs were isolated from 2-month-old (young) and 24-month-old rat (senescent) ventricles by the acid-guanidium-phenol-chloroform method. The first-strand synthesis of the cDNAs from each RNA was carried out with oligo-d(T) primers. The differential display screening was performed with three arbitrary primers and eight anchor primers, and the products were isolated on a 6% denaturing polyacrylamide gel. The bands showing differential expression were excised and subcloned into T-vector. We selected 19 upregulated clones and 66 downregulated clones in aged rat hearts. The differential expression of those candidate genes was confirmed by reverse Northern blot analysis. The selected genes were sequenced by dye-terminator methods. Among 31 clones, 15 clones were unknown. The known products included alpha-myosin heavy chain, cytochrome oxidase subunit, H+-transporting ATP synthase F0 complex subunit c isoform 3 (ATP5G3), and Na+-K+-Cl− cotransporter. The RT-PCR differential display method effectively identified genes differentially expressed in senescent hearts, and may be a useful tool for investigating factors responsible for age-related physiological changes.