Sirtuin 3 (SIRT3) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, which belongs to the Silent information regulator 2 (Sir2) family of histone deacetylases (sirtuin HDACs). The yeast Sir2 protein and its mammalian derivatives play a central role in epigenetic gene silencing, DNA repair and recombination, cell-cycle, microtubule organization, and in the regulation of aging. We have isolated and characterized the murine Sirt3 genomic sequence, which spans a region of 18,646 bp and which has one single genomic locus. Determination of the exon-intron splice junctions identified murine SIRT3 to be encoded by 7 exons ranging in size from 101 (exon 4) to 420 bp (exon 7). Characterization of the 5' flanking genomic region, which precedes the murine Sirt3 open reading frame, revealed a number of STATx, GATA and SP1 transcription factor binding sites. A CpG island was not detected. The 1,473-bp murine Sirt3 transcript has an open reading frame of 774 bp and encodes a 257-aa protein (cytoplasmic SIRT3) with a predictive molecular weight of 28.8 kDa and an isoelectric point of 5.82. Recently, a 1,406-bp murine SIRT3 splice variant that encodes a 334-aa mitochondrial precursor protein with a molecular weight of 36.6 kDa and an isoelectric point of 7.19 has been described. Fluorescence in situ hybridization analysis identified a single genomic locus for murine Sirt3 gene on chromosome 7F4 and which is neighbored by the Ric8 and PSMD13 genes. Our study brings light and a number of corrections and additions to previous reports on the genomic organization and the genomic sequence of murine Sirt3, which may be of importance in view of studies on potential genetic polymorphisms in relation to cellular respiration, metabolism, aging-related disease and cancer.