Medium length (500-200 bp) alternating purine-pyrimidine DNAs were prepared by sonication of synthetic polymers at low temperature. The products, and the hairpin structures derived from them after melting, were sufficiently small for high-resolution 31P NMR studies. Of the five sequences studied, two DNAs, poly(dG-dC).poly(dG-dC) and poly(dA-dU).poly(dA-dU), gave singlet 31P resonances, while three others, poly(dA-dT).poly(dA-dT), poly(dA-br5U).poly(dA-br5U), and poly(dI-dC).poly(dI-dC), exhibited two resolved signals of equal area. This indicates the existence of two distinct alternating phosphodiester backbone conformations for these latter three B-DNAs in solution. Controls of homopolymers, which were also prepared by sonication, showed only singlet 31P resonances. Of the alternating sequences DNAs, only sonicated poly(dG-dC).poly(dG-dC) exhibited a conformational transition to a high salt (greater than 2.5 M) form which exhibited two well-resolved 31P resonances of equal area. This indicates that the high salt form of poly(dG-dC).poly(dG-dC) also has an alternating backbone structure, and it is presumed to be a Z-DNA. These results indicate a general response of the DNA backbone conformation to alternating purine-pyrimidine base sequences but with a degree of sequence and environmental specificity which might have functional genetic significance.