A butyric acid pro-drug, pivaloyloxymethyl butyrate, AN-9, developed in our laboratory, was previously shown to act as a differentiation-inducing and an anti-cancer agent. In this study we have shown that both AN-9 and butyric acid caused a transient hyperacetylation of histones, which returned to basal levels after 6 and 12 hr, respectively. This activity precedes the induction of differentiation elicited by both agents. AN-9 induced acetylation of histones at a concentration one order of magnitude lower than butyric acid. Pre-treatment of the cells with esterase(s) inhibitors diminished the ability of AN-9 to inhibit proliferation and induce differentiation. The above suggests that the intracellular release of butyric acid fragment, from the pro-drug, is catalyzed by cellular esterase(s).