When chromatin is extracted from nuclease-digested nuclei by stepwise salt elution, two different classes of newly replicated chromatin can be distinguished. Nascent DNA eluted from nuclei under conditions of low to moderate ionic strength (0.1-0.3 M NaCl) exhibits nucleosomal periodicity and is found in particles which have the same electrophoretic mobility as bona fide H1- or high mobility group protein-containing mononucleosomes. Thus, factors believed to be involved with both the higher order coiling and transcriptionally active state of chromatin are rapidly complexed with newly synthesized DNA and may be retained on parental nucleosomes throughout replication. In contrast, approximately 40% of new DNA is resistant to extraction with solutions of moderate ionic strength. Most of this material is eluted from nuclei by 0.4-0.6 M NaCl. While bulk chromatin that is extracted by 0.4-0.6 M NaCl is organized into nucleosomes, most of the newly replicated "chromatin" from the same fractions lacks subunit structure, as determined by DNA size analyses in polyacrylamide gels, thereby distinguishing this nascent material from newly replicated chromatin eluted at lower ionic strength. Within 15 min all newly synthesized chromatin matures and exhibits the solubility and nucleosomal periodicity characteristics of bulk chromatin. The unusual properties of the "nonnucleosomal" fractions may reflect the structure of newly synthesized DNA prior to its assembly into nucleosomes.