In contrast to many other type II restriction endonucleases, EcoRV binds specifically to DNA only in the presence Mg2+. According to the co-crystal structure of an EcoRV-DNA complex, Mg2+ ion(s) bind to the active site of EcoRV liganded by Glu45, Asp74, and Asp90. Here we present experimental evidence suggesting that the EcoRV-DNA complex also interacts with Mg2+ ions at other sites: (i) We have prepared an EcoRV triple mutant, in which all acidic amino acids in the catalytic center are replaced by alanine. This mutant is catalytically inactive. It binds nonspecifically to DNA in the absence of Mg2+, whereas it binds specifically to DNA in the presence of Mg2+. This means that Mg2+ induces specific DNA binding in this mutant, although all Mg2+ ligands in the catalytic center are removed. Therefore, additional interactions between Mg2+ and the EcoRV-DNA complex probably occur at sites distinct from the catalytic center. (ii) We have measured the specific and nonspecific DNA binding constants of EcoRV and of the triple mutant in the presence and absence of Mg2+. Mg2+ reduces nonspecific binding by 3-4 orders of magnitude, presumably because Mg2+ ions bound to the DNA have to be released upon complex formation. In contrast, the specific binding of the wild-type enzyme and the triple mutant is increased in the presence of Mg2+. This result can only be explained if a Mg2+ ion binds to the specific EcoRV-DNA complex probably at a site distinct from the catalytic center.(ABSTRACT TRUNCATED AT 250 WORDS)