The biochemical and physicochemical properties of DNA oligomers containing phosphorodithioate linkages in various configurations were evaluated. Duplex stability studies, which were carried out by thermal denaturation analysis with complementary unmodified DNA, indicated a highly cooperative process similar to completely unmodified duplexes. Oligomers containing phosphorodithioate linkages were found to have reduced melting temperatures relative to unmodified duplexes, with the degree of Tm depression paralleling the percent phosphorodithioate composition of the oligomer. Relative to activation of RNase H, DNA oligomers containing up to 50% phosphorodithioate linkages were able to direct RNase H degradation with the same efficiency as unmodified DNA while those containing from 50 to 100% acted with somewhat reduced efficiency. At limiting concentrations, an oligomer containing alternating phosphorodithioate and phosphate linkages was able to direct RNase H degradation of the target RNA in an extended incubation, while an unmodified oligomer did not. The nuclease resistance of phosphorodithioate-containing oligomers was evaluated in HeLa cell nuclear and cytoplasmic extracts, in human serum, and with nucleases S1 and DNase I. Oligomers containing alternating phosphorodithioate and phosphate were highly resistant to degradation in all systems. However, oligomers having more than one unmodified linkage separating phosphorodithioates were degraded rapidly by DNase I, while demonstrating stability to degradation in all other systems tested. These results indicate that phosphorodithioate-containing DNA oligomers are highly nuclease-resistant, are able to form stable duplexes with complementary nucleic acid sequences, and efficiently direct RNase H degradation of target RNA.