Biological activities of four chemically synthesized trichostatin-related compounds, (R)-trichostatin A, (S)-trichostatin A, (R)-trichostatic acid, and (S)-trichostatic acid, were investigated. Assays of differentiation-inducing activity in Friend leukemia cells and G2-arresting activity in the cell cycle of normal rat fibroblast cells were used as monitoring systems for comparing the bioactivities of these compounds. The results clearly showed that both of the enantiomers of trichostatic acid had no activity in both the assay systems. In the case of (S)-trichostatin A, the antipode of naturally occurring trichostatin A, 50% effective concentrations were determined to be 50-70-fold higher than those of (R)-trichostatin A. The relationship between this ratio and the value of enantiomeric excess strongly suggests that (S)-trichostatin A is also biologically inactive. These results indicate that the absolute configuration and the presence of the hydroxamate group of trichostatin A are essential for its biological activity.