Z-DNA binding protein 1 (ZBP1) belongs to a family of proteins that contain the Zalpha domain, which binds specifically to left-handed Z-DNA and Z-RNA. Like all vertebrate proteins in the Zalpha family, it contains two Zalpha-like domains and is highly inducible by immunostimulation. Using circular dichroism spectroscopy and electrophoretic mobility shift assays we show that both Zalpha domains can bind Z-DNA independently and that substrate binding is greatly enhanced when both domains are linked. Full length ZBP1 and a prominent splice variant lacking the first Zalpha domain (DeltaZalpha) showed strikingly different subcellular localizations. While the full length protein showed a finely punctate cytoplasmatic distribution, ZBP1DeltaZalpha accumulated in large cytoplasmic granules. Mutation of residues important for Z-DNA binding in the first Zalpha domain resulted in a distribution comparable to that of ZBP1DeltaZalpha. The ZBP1DeltaZalpha granules are distinct from stress granules (SGs) and processing bodies but dynamically interacted with these. Polysome stabilization led to the disassembly of ZBP1DeltaZalpha granules, indicating that mRNA are integral components. Heat shock and arsenite exposure had opposing effects on ZBP1 isoforms: while ZBP1DeltaZalpha granules disassembled, full length ZBP1 accumulated in SGs. Our data link ZBP1 to mRNA sorting and metabolism and indicate distinct roles for ZBP1 isoforms.