Sister chromatids are tethered together by the cohesin complex from the time they are made until their separation at anaphase. The ability of cohesin to tether sister chromatids together depends on acetylation of its Smc3 subunit by members of the Eco1 family of cohesin acetyltransferases. Vertebrates express two orthologs of Eco1, called Esco1 and Esco2, both of which are capable of modifying Smc3, but their relative contributions to sister chromatid cohesion are unknown. We therefore set out to determine the precise contributions of Esco1 and Esco2 to cohesion in vertebrate cells. Here we show that cohesion establishment is critically dependent upon Esco2. Although most Smc3 acetylation is Esco1 dependent, inactivation of the ESCO1 gene has little effect on mitotic cohesion. The unique ability of Esco2 to promote cohesion is mediated by sequences in the N terminus of the protein. We propose that Esco1-dependent modification of Smc3 regulates almost exclusively the noncohesive activities of cohesin, such as DNA repair, transcriptional control, chromosome loop formation, and/or stabilization. Collectively, our data indicate that Esco1 and Esco2 contribute to distinct and separable activities of cohesin in vertebrate cells.