Abstract The reassociation kinetics of human DNA was studied, utilizing S1 nuclease digestion in aqueous dioxane and hydroxyapatite chromatography for isolating renatured DNA. The percentage of DNA reassociated at C 0t = 10 −3 was 5–7% and that at C 0t = 18 000 was about 85%, C 0t being the product of the molar concentration of DNA and the reassociation period in s. The shape of the amended reassociation curve was roughly that of a rectangular hyperbola. It showed pronounced differences from the curves obtained by direct hydroxyapatite chromatography of reassociated DNA. The S1 nuclease-dioxane procedure offered two advantages over the conventional method. It was applicable to the study of reassociation with high molecular weight DNA, and the reassociated DNA so obtained was devoid of low-melting strands. The analysis of the new data took into account the possible effects of the diploid condition on the reassociation rate of DNA, the source of the DNA used in this study being placental tissue. The new reassociation profile was compared to ideal second-order reassociation curves calculated for the human genome (2.5 · 10 9 nucleotide pairs), and for a genome twice this size, containing various proportions of single-copy sequences. The results showed that approximately 85–90% of the total DNA may consist of unique sequences. This estimate is considerably higher than those reported previously.