Cohesins functionally associate with CTCF on mammalian chromosome arms. Cohesins mediate sister chromatid cohesion, essential for chromosome segregation and DNA repair. They also regulate gene expression and enhancer-promoter interactions. Previous studies in yeast showed cohesin binding correlated with transcriptional activity, but in mammals, cohesin distribution is not driven by transcription. Instead, mammalian cohesins occupy a subset of DNase I hypersensitive sites, many of which contain sequence motifs resembling the consensus for CTCF, a DNA-binding protein with enhancer-blocking and boundary-element functions. CTCF is required for cohesin localization to these sites. Recruitment by CTCF suggests a rationale for noncanonical cohesin functions, as CTCF binding is sensitive to DNA methylation, allowing cohesin positioning to integrate DNA sequence and epigenetic state. Cohesins bind to a subset of constitutive DNase I hypersensitive sites, including conserved noncoding sequences. CTCF and cohesins colocalize extensively in mammalian cells, and loss-of-function experiments show that CTCF largely determines cohesin localization. Cohesin recruitment by CTCF provides a mechanism for the selective positioning of cohesins on mammalian chromosome arms and explains the preference of cohesins for constitutive HSS without classical enhancer or promoter function. Cohesin binding is compatible with locus remodeling and high-level expression. Cohesin sites are conserved between species, and cohesin binding is compatible with CTCF-dependent insulator function. CTCF binding is influenced by DNA methylation, explaining the appearance of novel cohesin sites after 5-Aza-cytidine treatment. Cohesin recruitment by CTCF links DNA sequence and epigenetic state. The study shows that cohesin positioning on mammalian chromosome arms is determined by local sequence context and chromatin structure, with a preference for DNase I hypersensitive sites and conserved noncoding sequences. CTCF is required for cohesin localization to these sites, and cohesin binding is compatible with CTCF-dependent insulator function. The findings provide a mechanism for the positioning of cohesins on mammalian chromosome arms and a rationale for noncanonical cohesin functions.Cohesins functionally associate with CTCF on mammalian chromosome arms. Cohesins mediate sister chromatid cohesion, essential for chromosome segregation and DNA repair. They also regulate gene expression and enhancer-promoter interactions. Previous studies in yeast showed cohesin binding correlated with transcriptional activity, but in mammals, cohesin distribution is not driven by transcription. Instead, mammalian cohesins occupy a subset of DNase I hypersensitive sites, many of which contain sequence motifs resembling the consensus for CTCF, a DNA-binding protein with enhancer-blocking and boundary-element functions. CTCF is required for cohesin localization to these sites. Recruitment by CTCF suggests a rationale for noncanonical cohesin functions, as CTCF binding is sensitive to DNA methylation, allowing cohesin positioning to integrate DNA sequence and epigenetic state. Cohesins bind to a subset of constitutive DNase I hypersensitive sites, including conserved noncoding sequences. CTCF and cohesins colocalize extensively in mammalian cells, and loss-of-function experiments show that CTCF largely determines cohesin localization. Cohesin recruitment by CTCF provides a mechanism for the selective positioning of cohesins on mammalian chromosome arms and explains the preference of cohesins for constitutive HSS without classical enhancer or promoter function. Cohesin binding is compatible with locus remodeling and high-level expression. Cohesin sites are conserved between species, and cohesin binding is compatible with CTCF-dependent insulator function. CTCF binding is influenced by DNA methylation, explaining the appearance of novel cohesin sites after 5-Aza-cytidine treatment. Cohesin recruitment by CTCF links DNA sequence and epigenetic state. The study shows that cohesin positioning on mammalian chromosome arms is determined by local sequence context and chromatin structure, with a preference for DNase I hypersensitive sites and conserved noncoding sequences. CTCF is required for cohesin localization to these sites, and cohesin binding is compatible with CTCF-dependent insulator function. The findings provide a mechanism for the positioning of cohesins on mammalian chromosome arms and a rationale for noncanonical cohesin functions.