Senescence marker protein-30 (SMP30) gene expression declines during aging in mouse liver. SMP30 also plays a role as Ca2+-binding protein localized in cytosol of hepatocytes. To elucidate the molecular mechanism of regulation of SMP30 gene expression we have cloned its gene promoter and carried out DNA-protein interaction analyses by DNase I footprinting and electrophoretic mobility shift assay. We have identified a total of eight nuclear factor binding sites within 0.8 kb upstream of transcription start site. Three of these sites are novel DNA sequences with no homology to the existing transcription factor binding site database. Interaction of nuclear factors to these novel cognate sites are DNA sequence-specific. The other five sites correspond to binding sites of known transcription factors, Sp1, AP2, CCAAT box, Lyf-1, and GATA-1. Coordinated orchestration of these factors may contribute to regulation of SMP30 gene expression.