The cellular redox state is associated with major cellular processes including differentiation, transformation, and apoptosis. Glutaredoxin 2 (Grx2) is a mitochondrial oxidoreductase suggested to play a critical role in protection against apoptotic stimuli. An alternative Grx2 transcript variant encoding a nonmitochondrial protein (Grx2b) was proposed before, but no data was available on the expression of this isoform. We have systematically investigated the expression of Grx2 transcript variants in human tissues and transformed cell lines. The transcript variant encoding mitochondrial Grx2 (Grx2a) was found to be ubiquitously expressed, emphasizing the general importance of the protein for mitochondrial redox homeostasis. In addition, we confirmed the previously suggested isoform Grx2b and identified a new third isoform (Grx2c) derived from alternative splicing of the Grx2b-encoding transcript. In normal tissue expression of both Grx2b and Grx2c was restricted to testes, but additionally we were able to demonstrate transcripts in various cancer cell lines. Both Grx2b and Grx2c are enzymatically active, but only Grx2c can complex the regulatory iron-sulfur cluster described for Grx2a. Expression of GFP fusion proteins suggested a cytosolic and nuclear localization of both Grx2b and Grx2c. Our findings provide the first evidence for functions of Grx2 outside mitochondria.