An assay which measures polydeoxyribonucleates containing covalently linked complementary sequences is described. It is based on the fluorescence enhancement of ethidium bromide bound to bihelical polydeoxyribonucleates. This dependence of fluorescence enhancement upon bihelicity has been further documented. Synthetic, defined polymers, such as d(A)n∙d(T)n, d(AT)n∙d(AT)n, d(TC)n∙d(GA)n, and d(TTG)n∙d(CAA)n, have been studied, as well as naturally occurring DNAs. At moderate ionic strengths, the synthetic polymers will re-anneal following heat denaturation. Their facility to renature is due to the self-complementarity between strands throughout their lengths. This facility is lost at very low ionic strengths (Γ/2 < 0.003 M), where ionic repulsion prevents the formation of "nucleation" sites for annealing. However, a covalent linkage between strands provides a nucleation site for renaturation. The fluorescence seen with ethidium bromide after heating and cooling such polymers is then a measure of DNA which has covalently linked complementary sequences. By this assay, natural DNAs appear to contain short, relatively unstable bihelical regions following heat denaturation and cooling, in agreement with models based on other physical measurements.