By using a series of formalin concentrations we have found that high aldehyde levels in the fixation buffer of Miller spreads are correlated with the appearance of nonnucleosomal stretches in newly replicated chromatin of embryos from Drosophila metanogaster. These nucleosome-free gaps are found O-500 nm behind the replication fork and do not correspond to naked DNA. The analysis of the distribution of nucleosome-free gaps on newly replicated DNA has revealed some structural details about the maturation of nucleosomes and provides direct evidence that parental nucleosomes have an altered structure at the replication fork. Finally, these stretches of nonnucleosomal chromatin are located in a trans disposition inside the active replicon, although there exists a considerable variability. © 1995 Academic Press, Inc.