A laboratory scale procedure developed for the purification of EcoRI restriction endonuclease was applied to two different Escherichia coli strains, E. coli 294 and E. coli M5248, which are genetically modified to overproduce the enzyme. The purification method consisted of three successive chromatographic steps including phosphocellulose and hydroxyapatite columns and further fractionation in a second phosphocellulose column. It a as shown that the second phosphocellulose separation can be omitted in the case of E. coli 294. Quality control tests indicated enzyme preparations free of contaminants and endo- or exo-nucleases. The yields obtained at the final stage of the purification were 1.3x10(5) U/g cells for E. coli M5248 and 3.3x10(6) U/g cells for E. coli 294.