T his study shows that tritiated thymidine labeled DNA prepared from mammalian cells by the Marmur technique is a pure preparation of nucleic acid that is composed essentially of two populations of molecules. One molecular population consists of primarily double-standed nucleic acid, while the other population is of double-stranded nucleic acid with significant single-stranded regions. The double-stranded DNA with single-stranded regions can, depending upon the length of the single strand, behave as "native" DNA or "denatured" DNA on methylated albumin kieselguhr (MAK) column chromatography, Using MAK chromatography we have separated the DNA into a saltelutable fraction composed of primarily double-stranded molecules and an alkaline-elutable fraction containing double-stranded nucleic acid with variable length, single-stranded regions. Endonuclease enzyme removal of the single-stranded regions from the alkaline fraction DNA yield nucleic acid that behaves identically to the salt elutable DNA. Exonuclease removal of the single-stranded regions suggests they are located primarily at the ends of the molecules. Our data show that the alkaline-elutable DNA differs from salt-elutable DNA only in that the former has significant single-stranded regions. Sera of patients with systemic lupus erythematosus (SLE) selected for anti-DNA by hemagglutination bind significantly less to the alkaline fraction DNA than the sale fraction DNA. This difference in binding clearly does not represent simply an affinity for double-stranded vs. single-stranded nucleic acid since the alkaline fraction DNA contains predominately double-stranded nucleic acid. A model for antibody-DNA binding is suggested from the present data and information contained in the literature.