Histone deacetylase (HDAC) inhibitors are powerful tools in understanding epigenetic regulation and have proven especially promising for the treatment of various cancers, but the discovery of potent, isoform-selective HDAC inhibitors has been a major challenge. We recently developed a cyclic alpha(3)beta-tetrapeptide scaffold for the preparation of HDAC inhibitors with novel selectivity profiles ( J. Am. Chem. Soc. 2009 , 131 , 3033 ). In this study, we elaborate this scaffold with respect to side chain diversity by synthesizing one-bead-one-compound combinatorial libraries of cyclic tetrapeptide analogues and applying two generations of these focused libraries to the discovery of potent HDAC ligands using a convenient screening platform. Our studies led to the first HDAC6-selective cyclic tetrapeptide analogue, which extends the use of cyclic tetrapeptides to the class II HDAC isoforms. These findings highlight the persistent potential of cyclic tetrapeptides as epigenetic modulators and possible anticancer drug lead compounds.