One polypeptide, designated S, confers sequence-specificity to the multisubunit type I restriction enzymes. Two families of such enzymes, K and A, include members that recognize diverse, bipartite, target sequences. The S polypeptides of the K family, while having areas of near identity, also contain two extensive regions of variable sequence. We now show that one of these, comprising the N-terminal 150 amino acids, specifies recognition of one component of the bipartite target sequence. We have determined the sequence recognized by EcoE, a member of the A family. This sequence, 5'GAG(N7)ATGC, has the trinucleotide GAG in common with EcoA and with StySB of the K family. We determined the nucleotide sequences of the S genes of EcoA and EcoE, and compared their predicted amino acid sequences with each other and with those of the five members of the K family. There is no general sequence similarity between families, but the domain of the S polypeptide of StySB, which specifies GAG, shows nearly 50 per cent identity with the amino variable region of the S polypeptides of EcoA and EcoE. A complex domain that recognizes and directs methylation of GAG is therefore common to enzymes of generally dissimilar amino acid sequence.