In situ digestion experiment using restriction enzyme applied to fixed human chromosome brought the following results; First, Asn I and Dra I that recognized AT-rich sequence produced C-like band patterns and telomere bands which are the distinctive feature of R-like band patterns. Therefore the existence of recognition base sequence greatly influenced for the action of restriction enzyme on fixed chromosome. Second, DNA loss had decisive influence upon C-like band pattern produced by restriction enzyme. But chromatin comformational change seems to have acted upon G-like band pattern. Third, analysis of the result of electrophoresis showed that among DNA fragments separated from a chromosome by restriction enzyme, some DNA fragments that had large size and were not extracted under the influence of non-histone protein were remained on fixed chromosome. Fourth, human constitutive heterochromatin turned out to be heterogeneous by satellite DNA of highly repeated sequence. In conclusion, there's no doubt that DNA loss plays the most important role in the formation of bands by restriction enzyme and that DNA loss depends on the frequency of recognition sequence of restriction enzyme.