The FK228 and spiruchostatin bicyclic depsipeptide natural products are among the most potent histone deacetylase (HDAC) inhibitors known. Although FK228 is in advanced clinical trials, the complexity of the natural products has precluded mechanistic studies and the discovery of structure−activity relationships. By total synthesis, we have prepared the first depsipeptide analogues. Our results prove that the dehydrobutyrine residue in FK228 is not essential, and other residues can be substituted without loss of HDAC inhibitory activity. Conformational restriction by the macrocyclic scaffold is important, as a linear peptide was inactive. The intramolecular disulfide formed with a cysteine side chain can be removed provided the zinc-binding thiol is protected to ensure good cellular availability. Like the natural products, the analogues are selective against class I isoforms, with nanomolar inhibition of class I HDAC1 and significantly less potency against class II HDAC6.