Protein acetylation, catalyzed by the opposing activities of histone deacetylases (HDAC) and histone acetyltransferases, is now recognized to be an important epigenetic modulator of gene transcriptional activity and cell function. As a result of the intense search for HDAC inhibitors (HDACi) during the past fifteen years, a large number of structurally divergent classes with variable potencies and isoenzyme selectivities have been identified. They occupy an important and promising position in a number of therapeutic areas. Several HDACi are under clinical evaluation as tumor cell-selective chemotherapeutics, and show great promise for the treatment of inflammatory disorders, neurodegenerative diseases, protozoal and latent viral infections, and (fibro)proliferative disorders. Recently, it was discovered that they might be used as enhancers of differentiation in stem cell therapy, and as medium supplements that stabilize the phenotype of primary cells in culture. Next to biological activity, the pharmaceutical potential of a compound is also dependent on the adequate translation of in vitro potency into in vivo efficacy whilst maintaining an acceptable safety profile. Therefore, this review will not only address the formerly mentioned applications, but will also deal with the pharmacokinetic and toxicological properties of currently available HDACi. Several compounds exert potent activities in vitro, but have been shown to be of limited therapeutic value due to rapid biotransformation, and thus poor in vivo bioavailability. The first attempts to improve the metabolic properties of HDACi have been made and will be discussed. In contrast to conventional chemotherapeutics, HDACi exert no drastic side effects at therapeutically effective doses. Although a bulk effect on histone acetylation is observed, HDACi display a remarkable tumor cell-selective toxicity. The mechanisms underlying these cell type-dependent differences in sensitivity to HDACi-mediated effects, however, remain largely elusive.