The phosphite triester method was adapted for automated synthesis of phosphate (backbone)-modified analogues of DNA. The use of phosphoramidite reagents to achieve substitution of the nonequivalent (diastereotopic) oxygens of an internucleotide phosphate linkage is nonselective, and leads to nonequivalent (diastereomeric) strands of DNA with R _p and S _p absolute configurations at the chiral, modified phosphorus position. Indications of the scope and limitations of the use of reversed-phase HPLC to separate these diastereomers have been obtained through studies of backbone-modified DNA analogues having phosphorothioate (P-S), phosphotriester (P-OR), and alkanephosphonate (P-R) linkages. Incorporation of these modifications in the self-complementary octanucleotide d(GGAATTCC) led to separable diastereomers, which form nonequivalent R _p · R _p and S _p · S _p duplexes. A combination of chemical and nuclear Overhauser effect NMR spectroscopic methods was developed to assign unambiguously, for the first time, the absolute configurations at phosphorus in these prototypal cases. The effects of backbone ethylation on DNA structure and dynamics were evaluated by NMR methods. Modified duplexes were used to probe for proposed phosphate contacts for EcoRI endonuclease, and to define, in concert with base-modified analogues, a recognition site for monoclonal anti-native DNA autoantibody.