The 3 A electron density map of a co-crystalline recognition complex between EcoRI endonuclease and the oligonucleotide TCGCGAATTCGCG reveals that a tight, complementary interface between the enzyme and the major groove of the DNA is the major determinant of sequence specificity. The DNA contains a torsional kink and other departures from the B conformation which unwind the DNA and thereby widen the major groove in the recognition site.