Restriction endonuclease EcoRV has been reported to be unable to distinguish its specific DNA site, GATATC, from non-specific DNA sites in the absence of the catalytic cofactor Mg2+, and thus to exercise sequence specificity solely in the catalytic step. In contrast, we show here that under appropriate conditions of pH and salt concentration, specific complexes with oligonucleotides containing the GATATC site can be detected by either filter-binding or gel-retardation. Equilibrium binding constants (K(A)) are easily measured by both direct equilibrium and equilibrium-competition methods. The preference for "specific" over "non-specific" binding at pH 7 in the absence of divalent cations is about 1000-fold (per mole of oligonucleotide) or 12,000-fold (per mole of binding sites). Ethylation-interference footprinting shows that the "specific" complex includes strong contacts to the phosphate groups GpApTpApTC. Specific DNA binding is strongly pH-dependent, decreasing about 15-fold for each increase of one pH unit above pH 6, but non-specific binding is not; thus, binding specificity decreases with increasing pH. Gel retardation and filter-binding at pH < or = 7 yield essentially identical values of K(A) for specific-site binding, but at pH > 7 gel retardation significantly underestimates K(A). Specific-site binding is stimulated about 700-fold by Ca2+ (not a cofactor for cleavage), but with non-cleavable 3'-phosphorothiolate and 4'-thiodeoxyribose derivatives whose response to Ca2+ is similar to that of the parent oligonucleotide, Mg2+ stimulates binding only fourfold and twofold, respectively. Thus, binding specificity is not dramatically enhanced by Mg2+. Taking into account discrimination in binding and in the first-order rate constant for phosphodiester bond scission, the overall discrimination exercised against the incorrect site GTTATC is about 10(7)-fold. EcoRV endonuclease is thus not a "new paradigm" for site-specific interaction without binding specificity, but like other type II restriction endonucleases achieves sequence specificity by discriminating both in DNA binding and in catalysis.