OBJECTIVE To determine and quantify the differences produced in chromatin structure after selective in situ DNA digestion with restriction endonuclease (RE). METHODS Chromatin fiber structure from a murine cell line was analyzed under light and electron microscopy before and after in situ digestion with AluI, HinfI and HaeIII using digital image analysis (DIA). The proposed DIA-based method entails the generation of a binary image and a skeleton characteristic of the chromatin fiber. Morphologic features (surface, perimeter, shape, number of triple points and Euler number) of the chromatin structure can then be quantified from the digitized images. The results of these experiments were compared with those obtained from direct digestion of naked DNA using the same endonucleases in an attempt to correlate the distribution of restriction sites, according to the DNA fragment size obtained, with the extent of chromatin disorganization after RE in situ digestion. RESULTS Homologous chromatin fiber regions are differentially affected by the action of each RE, although they are indistinguishable under light microscopy. CONCLUSIONS The proposed analytical routine constitutes a valuable tool for uncovering subtle alterations in the structure of chromatin fiber that has been modified under different experimental conditions.