Electron microscopy (EM) of whole mounted mouse chromosomes, light microscopy (LM), and agarose gel electrophoresis of DNA were used to investigate the cytological effect on chromosomes of digestion with the restriction endonucleases (REs) AluI, HinfI, HaeIII and HpaII. Treatment with AluI produces C-banding as seen by LM, cuts DNA into small fragments, and reduces the density of centromeres and disperses the chromatin of the arms as determined by EM. Treatment with HinfI produces C-banding, cuts DNA into slightly larger fragments than does AluI and increases the density of centromeres and disperses the fibres in the chromosomal arms. Exposure to HaeIII produces G- + C-banding, cuts the DNA into large fragments, and results in greater density of centromeres and reduced density of arms. Finally HpaII digestion produces G-like bands, cuts the DNA into the largest fragments found and results in greater density of centromeres and the best preservation of chromosomal arms detected by EM. These results provide evidence for: (1) REs producing identical effects in the LM (AluI and HinfI) produce different effects in the EM. (2) All enzymes appear to affect C-bands but while REs such as AluI reduce the density of these regions, other enzymes such as HpaII, HaeIII or HinfI increase their density. Conformational changes in the chromatin could explain this phenomenon. (3) The appearance of chromosomes in the EM is related to the action of REs on isolated DNA. The more the DNA is cut by the enzyme, the greater the alteration of the chromosomal ultrastructure.