The restriction endonuclease EcoRI hydrolyzes DNA to a greater number of fragments in the presence of glycerol than under normal conditions. This enzyme begins to work by the so-called EcoRI-type of restriction when glycerol concentration reaches 50%. The EcoRI activity appeared in experiments only when the ionic strength of the solution was decreased and pH of the solution was increased. However, under such extreme conditions the enzyme was quickly inactivated and it was difficult to obtain reproducible results especially for hydrolysis of the high-molecular DNA. The suggested conditions for the EcoRI activity permit to obtain reproducible results, this being practically equivalent to discovery of the new restriction endonuclease.