Recent studies have shown that the protein CTCF, which plays an important role in insulation and in large-scale organization of chromatin within the eukaryotic nucleus, depends for both activities on recruitment of the cohesin complex. We show here that the interaction of CTCF with the cohesin complex involves direct contacts between the cohesin subunit SA2 and specific regions of the C-terminal tail of CTCF. All other cohesin components are recruited through their interaction with SA2. Expression in vivo of CTCF mutants lacking the C-terminal domain, or with mutations at sites within it required for SA2 binding, disrupts the normal expression profile of the imprinted genes IGF2-H19 and also results in a loss of insulation activity. Taken together, our results demonstrate that specific sites on the C terminus of CTCF are essential for cohesin binding and insulator function. The only direct interaction between CTCF and cohesin involves contact with SA2, which is external to the cohesin ring. This suggests that in recruiting cohesin to CTCF, SA2 could bind first and the ring could assemble subsequently.