Poly(ADP-ribose)glycohydrolase (PARG) is the major enzyme capable of rapidly hydrolyzing poly(ADP-ribose) (PAR) formed by the diverse members of the PARP enzyme family. This study presents an alternative splice mechanism by which two novel PARG protein isoforms of 60 kDa and 55 kDa are expressed from the human PARG gene, termed hPARG60 and hPARG55, respectively. Homologous forms were found in the mouse (mPARG63 and mPARG58) supporting the hypothesis that expression of small PARG isoforms is conserved among mammals. A PARG protein of approximately 60 kDa has been described for decades but with its genetic basis unknown, it was hypothesized to be a product of posttranslational cleavage of larger PARG isoforms. While this is not excluded entirely, isolation and expression of cDNA clones from different sources of RNA indicate that alternative splicing leads to expression of a catalytically active hPARG60 in multiple cell compartments. A second enzyme, hPARG55, that can be expressed through alternative translation initiation from hPARG60 transcripts is strictly targeted to the mitochondria. Functional studies of a mitochondrial targeting signal (MTS) in PARG exon IV suggest that hPARG60 may be capable of shuttling between nucleus and mitochondria, which would be in line with a proposed function of PAR in genotoxic stress-dependent, nuclear-mitochondrial crosstalk.