Two restrictases SsoI and SsoII, belonging to the enzymes of restriction of the class II, were isolated from a strain of dysenteric bacteria. The structure of the site sensitive to SsoI and SsoII was studied after fragmentation of testor DNA as well as by means of direct determination of nucleotide sequence. SsoI was shown to be an isoschizomer of the EcoRI restrictase from E. coli. Restrictase SsoII proved to be a new enzyme, which hydrolyzed the sequence 5' ...CCNGG.. 3' and was distinct from the known restrictases as shown by studies of the type of DNA hydrolyzed. A three-step procedure is developed for isolation of SsoII restrictase involving the consecutive chromatography on Blue Sepharose, phosphocellulose PII and phenyl-Sepharose. Restrictase SsoI and EcoRI were isolated by means of isoelectrofocusing using ampholines.