Two linear minus-strand viral DNAs, disparate in size by 10 nucleotides, were isolated from duck hepatitis B virus infected tissues and observed to migrate differently in nondenaturing agarose gels. We examined this phenomenon using both synthetic and cloned viral DNAs and discovered that distinct, circularly permuted, linear isomers of single-stranded DNA could have slightly different electrophoretic mobilities under nondenaturing conditions. This finding reveals a novel feature for consideration in assessing the conformations of native or renatured single-stranded nucleic acids. The study also suggests that the virion-derived minus-strand DNAs of the avihepadnaviridae may necessarily possess a minimal secondary structure.