DNA extracted from isolated hepatic nuclei of rats at different aged (1 h, 6 and 30 days of life) has been characterized by (i) melting temperature, (ii) buoyant density, (iii) thermal denaturation on hydroxyapatite and (iv) nucleoside composition. The melting midpoint (Tm) determined spectrophotometrically in 0.1 X SSC (0.15 M NaCl/0.0015 M sodium citrate) is 71.9 +/- 0.4 for 1-h-old rats and decreases to 70.7 +/- 0.3 in 6-day-old animals. The buoyant densities of DNAs determined by CsCl on both native and alkaline-denaturated and reneutralized DNA were also found to decrease with age. Hydroxyapatite thermal denaturation of sonicated DNA confirmed the significant difference between the Tm values of 1-h-old and 6-day-old rats (86.5 +/- 0.5 and 85.2 +/- 0.1, respectively). The possibility that these differences in Tm values could be due to an increase in methyl bases, has been ruled out by the finding that the amount of [3H]methyl incorporated in relation to the DNA synthesis is constant at these two ages. The alternative possibility of a change in base composition has been tested by the chromatographic analysis of nucleosides. The dG + dC content is 0.433 +/- 0.003 in 1-h-old rats and decreases to 0.411 +/- 0.002 and to 0.403 +/- 0.005 in 6-day- and 30-day-old rats, respectively. The physiological significance of the different base composition is discussed in relation to the possibility that specific DNA sequences are synthesized during the non-premitotic synthesis which has been found to take place during the first 6 days of life.