Background: How much DNA remains in mouse hepatocyte nuclei after extended chromatin fiber (ECF) formation or whether this content varies within the nuclear population is not known. This information could be relevant to understanding chromatin extensibility as related to chromatin organization, possibly associated with variable nuclear activities in hepatocytes. Methods: A protocol for ECF formation under the gravity action, image analysis of Feulgen-stained unfixed mouse hepatocyte remnants, and DAPI fluorescence were used. Results: Areas, shape, Feulgen-DNA amounts, and chromatin texture were affected in unfixed, lysed nuclei. The Feulgen-DNA values in nuclear remnants represented similar to 37% of the content in fixed, nonlysed nuclei in terms of median values; the coefficient of variation of Feulgen-DNA values in the nuclear remnants was much higher than those in controls. Enhancement in DAPI fluorescence was evident in chromocenters of the fixed nuclei and in remnants and some ECF granules of the unfixed, lysed nuclei. Conclusions: The DNA content of the nuclear remnants was much more variable than that assumed from known variability in hepatocyte ploidy degrees. The variable constraint to chromatin extrusion from hepatocyte nuclei is, hypothesized to depend on variable chromatin organization with possible involvement of nuclear matrix association, transcriptional, activities, and AT-rich DNA-containing heterochromatin. (c) 2006 International Society for Analytical Cytology.69A1111061113