Z-DNA is a left-handed helical form of DNA in which the double helix winds to the left in a zigzag pattern. DNA containing alternating purine and pyrimidine repeat tracts have the potential to adopt this non-B structure in vivo under physiological conditions, particularly in actively transcribed regions of the genome. Z-DNA is thought to play a role in the regulation of gene expression; Z-DNA is also thought to be involved in DNA processing events and/or genetic instability. For example, Z-DNA-forming sequences have the potential to enhance the frequencies of recombination, deletion, and translocation events in cellular systems. Although the biological function(s) of Z-DNA and related Z-DNA-binding proteins are not fully understood, accumulating experimental and clinical evidence support the idea that this non-B DNA conformation is involved in several important biological processes and may provide a target for the prevention and treatment of some human diseases. In this review, we discuss the properties of Z-DNA, proteins that are known to bind specifically to Z-DNA, and potential biological functions of this non-canonical DNA structure.