Restriction endonuclease, EcoR1 cleaves the DNA sequence (5′-3′)G↓¯A-A-T-T-C(3′-5′)C-T-T-A-A↑¯C at the Points indicated¹. Under certain conditions, EcoR1^* activity is observed when (5′-3′)N1↓¯A-A-T-T-N2(3′-5′)N3-T-T-A-A↑¯N4 is cut². Mg²+ is required for both activities.We find that in addition to binding to the above sites, EcoR1 will also bind, although less strongly, to DNA containing neither site. Methyl acetimidate, which reacts specifically with lysine residues, inactivates the enzyme. This specific effect can be prevented by SV40 DNA and lambda DNA which contain EcoR1 and EcoR1^* sites, by øX174 DNA, which has only EcoR1^* sites and also by Polyd(AT) and polyd(GC) containing neither site. Protection occurs in the absence or presence of magnesium.The significance of this non-specific binding, both for the use and mechanism of EcoR1 will be discussed.