The capacity of six sequences with different numbers and orientations of A.T pairs flanked by alternating C.G pairs to adopt left-handed structures was evaluated in recombinant plasmids. A series of synthetic oligodeoxynucleotides were cloned into the BamHI site of pRW790, a small plasmid (approximately 2 kilobases) prepared especially for conformational studies of this type. Supercoil relaxation studies by two-dimensional gel electrophoresis on topoisomers of each plasmid revealed the energetics and structures of the left-handed helices. Also, the presence of supercoil-induced altered DNA conformations within the inserts of topoisomer populations of the plasmids was detected by reaction with S1 nuclease followed by restriction mapping of the cleavage sites. We conclude that consecutive T.A base pairs, whether alternating (TATA) or contiguous (TTTT), can adopt a left-handed conformation (presumably Z) when flanked by reasonably short runs of alternating (C-G)n (n = 3-5). Thus, these results substantially broaden the range of DNA sequences that can adopt left-handed Z conformations.