Technical Abstract: SIRT3 is one of the seven mammalian sirtuin homologs of the yeast SIR2 gene. SIRT3 possesses NAD(+)-dependent protein deacetylase activity. Recent studies indicate that the murine SIRT3 gene expresses different transcript variants, resulting in three possible SIRT3 protein isoforms with various lengths at the N-terminus: M1 (aa 1-334), M2 (aa 15-334), and M3 (aa 78-334). The transcript variants 1 and 3 can only produce M3 protein, while M1 and M2 proteins are translationally initiated from different in-frame ATG sites in transcript 2. Here we report that three transcript variants of the mouse SIRT3 gene are broadly expressed in various mouse tissues. By expressing these SIRT3 isoforms in HEK293 cells through transient transfection, we confirmed recent reports that two longer murine SIRT3 proteins (M1 and M2) are targeted to mitochondria with higher efficiency than the shorter M3 isoform. Additionally, the M1 and M2 proteins are processed into a mature form. Using Edman degradation we identify Ile38 (majority) or Val42 as the N-terminal amino acid of the mature M1 isoform, and Met78 or Val79 as the N-terminal amino acid of the M3 isoform. Interestingly, we found that even upon mutation of the M2 ATG site in the M1 cDNA, a processed mature protein could still be produced. In terms of deacetylase activity, we found that although only the mature protein derived from M1 or M2 proteins were active against acetylated peptide substrates, all three forms had equal deacetylase activity towards a full-length native protein substrate, acetyl CoA synthetase 2.