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The fractionation of nucleic acids by chromatography on hydroxyapatite has been studied. The products, formed by annealing T2 DNA with complementary RNA, were separated in a linear gradient of phosphate buffer into three fractions containing, respectively, in their order of elution, single-stranded DNA, RNA complexed mainly with single-stranded DNA, and renatured DNA complexed with smaller amounts of RNA. By using annealing conditions which favour hybrid formation the complementary strand of DNA can be separated by a batchwise fractionation procedure.Chromatography on hydroxyapatite has previously been used for the separation of single-stranded DNA from native [1,2] or renatured [3,4] DNA, as well as for the fractionation of different types of RNA [I]. The elution conditions for different nucleic acids appears to be determined largely by their secondary structure [l]. A study of the properties of RNA-DNA complexes by fractionation on hydroxyapatite was undertaken. Several methods have previously been developed for the isolation of complexes between DNA and complementary RNA [5]. However, neither of these have aimed at the separation of the complementary strands of DNA.Separation of the complementary strands of DNA has been achieved by ultracentrifugation methods, most frequently aided by synthetic polynucleotides with specific affinity for one of the strands [6]. Kieselguhr coated with methylated serum albumin has also been used successfully [7]. The strand separation obtained by these methods depends on differences in the base composition of the two strands. The separation of the materials after RNA-DNA hybridization into a RNA-DNA complex fraction and a single-stranded DNA fraction offers an alternative method, since only minus strand DNA is capable of forming a complex with RNA. When annealing DNA with complementary RNA in solution, DNA renaturation and RNA-DNA complex formation are competing processes, both of second order. Formation of RNA-DNA complexes predominates when annealing DNA a t low concentrations with a large excess of complementary RNA Enzyme. Deoxyribonuclease (EC 3.1.4.5).[8]. The present communication describes experiments in which separation of the strands is achieved by hydroxyapatite chromatography of complexes formed during annealing conditions which favour hybrid formation. Some of the results presented in this paper have previously been reported [3]. MATERIALS AND METHODSPreparation of 32P-Labelled RNA from T2 Infected E. coli Escherichia coli B was grown under aeration in a synthetic salt-glucose medium with inorganic phosphate replaced by 0.08 Ol0 bacto-tryptone. The culture was infected with 10 phage T2 per cell a t a cell density of 4 x los cells/ml. The culture was poured into crushed ice and centrifuged 6 or 16 min after infection for the preparation of "early" or "late" RNA respectively. 0.2 mC 32P-labelled phosphate per 1200 ml of culture was added 2 to 2.5 min before harvesting. Extraction of the RNA was performed by lysing the cells in 0.5O/, sodium dodecylsulfate, followed by...