The type IC DNA methyltransferase M.EcoR124I is a trimeric enzyme of 162 kDa consisting of two modification subunits, HsdM, and a single specificity subunit, HsdS. Studies have been largely restricted to the HsdM subunit or to the intact methyltransferase since the HsdS subunit is insoluble when over-expressed independently of HsdM. Two soluble fragments of the HsdS subunit have been cloned, expressed and purified; a 25 kDa N-terminal fragment (S3) comprising the N-terminal target recognition domain together with the central conserved domain, and a 8.6 kDa fragment (S11) comprising the central conserved domain alone. Analytical ultracentrifugation shows that the S3 subunit exists principally as a dimer of 50 kDa. Gel retardation and competition assays show that both S3 and S11 are able to bind to HsdM, each with a subunit stoichiometry of 1:1. The tetrameric complex (S3/HsdM)2 is required for effective DNA binding. Cooperative binding is observed and at low enzyme concentration, the multisubunit complex dissociates, leading to a loss of DNA binding activity. The (S3/HsdM)2 complex is able to bind to both the EcoR124I DNA recognition sequence GAAN6RTCG and a symmetrical DNA sequence GAAN7TTC, but has a 30-fold higher affinity binding for the latter DNA sequence. Exonuclease III footprinting of the (S3/HsdM)2 -DNA complex indicates that 29 nucleotides are protected on each strand, corresponding to a region 8 bp on both the 3′ and 5′ sides of the recognition sequence bound by the (S3/HsdM)2 complex.