Electron microscopic (EM) analysis of DNA–DNA heteroduplexes permits the evaluation of sequence and organizational relationships among related genomic and subgenomic molecules. Formamide is used to dissociate adventitious intrastrand pairing during EM grid preparation, facilitating the measurement of both double-stranded and single-stranded DNA segments. There are two forms of RNA–DNA heteroduplexes. In an R-loop, the DNA remains duplexed, except where RNA has hybridized and has caused the displacement of its DNA counterpart, leading to the formation of a loop. In the RNA-single-stranded DNA heteroduplexes (R–D heteroduplexes), the DNA remains unpaired, except where RNA has hybridized. The composition of the RNA–DNA hybridization solution is narrowly dictated by the high concentration of formamide required. The concentration of NaCl should be as high as practicable to promote the maximal rate of annealing. © 1989, Elsevier Inc. All rights reserved.