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Summary Cohesin entraps sister DNAs within tripartite rings created by pairwise interactions between Smc1, Smc3, and Scc1. Because Smc1/3 ATPase heads can also interact with each other, cohesin rings have the potential to form a variety of sub-compartments. Using in vivo cysteine cross-linking, we show that when Smc1 and Smc3 ATPases are engaged in the presence of ATP (E heads), cohesin rings generate a “SMC (S) compartment” between hinge and E heads and a “kleisin (K) compartment” between E heads and their associated kleisin subunit. Upon ATP hydrolysis, cohesin’s heads associate in a different mode, in which their signature motifs and their coiled coils are closely juxtaposed (J heads), creating alternative S and K compartments. We show that K compartments of either E or J type can entrap single DNAs, that acetylation of Smc3 during S phase is associated with J heads, and that sister DNAs are entrapped in J-K compartments., Graphical Abstract, Highlights • Smc1 and Smc3 ATPase heads adopt an engaged (E) and a juxtaposed (J) state in vivo • Smc ATPase heads delimit an Smc (S) and a kleisin (K) compartment • Single DNA molecule can be entrapped inside K compartments of either E or J type • Sister DNAs are entrapped in J-K compartments with J-head Smc3 being acetylated, Sister chromatid cohesion is thought to be conferred by sister DNA entrapment within the cohesin ring. Chapard et al. show that cohesin’s Smc heads interact with each other in two different ways in vivo, allowing sister DNA entrapment between juxtaposed ATPase heads and kleisin of the cohesin complex.