Extensive studies of DNA secondary structure during the past decade have shown that DNA is a dynamic molecule, whose structure depends on the underlying nucleotide sequence and is influenced by the environment and the overall DNA topology. Three major non-B-DNA structures have been described (Z-DNA, triplex DNA and cruciform DNA) which are stabilized by unconstrained negative supercoiling and can be formed under physiological conditions. In this essay we summarize the DNA primary structure features that are pertinent to the formation of these conformers and present data concerning the occurrence of these sequences in the eukaryotic genome. The evidence in favor of the existence of these unusual DNA structures in vivo is discussed. The effect of alternative non-B-DNA structures on the way DNA is organized in chromatin is considered, and this is followed by evaluation of the data relating these structures to eukaryotic transcription. Some possible mechanisms by which the effect of non-B structures on transcription might be exerted are proposed.