DNA strands with appropriate sequences of dA and dT can form a stable duplex in which the two strands adopt a parallel (ps) instead of the conventional antiparallel (aps) orientation. Four 25-nt dA .cntdot. dT-containing deoxyoligonucleotide (D1-4) were synthesized. D1 has the sequence 5''-dA10TA2T4A3TAT3-3''. Viewed with the same polarity, D2, D3, and D4 are the complement, inverted complement, and inverse of D1, respectively. The two combinations D1 .cntdot. D3 and D2 .cntdot. D4 form conventional antiparallel duplexes (aps-D1 .cntdot. D3, aps-D2 .cntdot. D4). D1 .cntdot. D2 and D3 .cntdot. D4, however, constitute stable parallel-stranded duplexes (ps-D1 .cntdot. D2, ps-D3 .cntdot. D4), as established by various criteria including the following: (i) The electrophoretic mobilities of ps-D1 .cntdot. D2 and ps-D3 .cntdot. D4 are similar to those of the antiparallel-stranded duplexes. (ii) The ultraviolet absorption and circular dichroism spectra of the ps duplexes are indicative of a base-paired structure, but differ systematically from those of the aps helices. (iii) Similar salt-dependent thermal transitions are observed for the four duplexes, but the melting temperatures of the ps molecules are lower by 13-18.degree. C.