This method provides a simultaneous spectral determination for both base composition and base concentration of DNA. Spectra of standard deoxynucleotide mixtures (adenylate with thymidylate and guanylate with cytidylate), hydrolyzed in 1 N acid, are directly proportional to spectra for DNA treated similarly. The significant products from hydrolysis are free purine bases and intact pyrimidine deoxynucleotides. There is a linear relation between the sum of the chosen differential absorbancies of the standard spectra and base composition. The base concentration of DNA is proportional to base concentration of the standards at an isosbestic point. The determinations are linear for base compositions of 0–100 mole % and for base concentrations of 0–100 μ M. Analyses of various DNA samples of animal and bacterial origin illustrate the practical application of the method. Literature values for base composition and phosphorus analyses for base concentration furnish comparisons with spectrally determined values as a test for accuracy. The method is not applicable to single-chain DNA's or to DNA's having unusual bases in high proportion.