Alkyl esters of (RS)-3-(adenin-9-yl)-2-hydroxypropanoic acid (AHPA) were shown recently to be broad spectrum antiviral agents (De Clercq E and Holy A, J Med Chem 28: 282-287, 1985). It was postulated that these alkyl esters function as prodrugs by undergoing hydrolysis catalyzed by cellular esters to AHPA, a known inhibitor of S-adenosyl-l-homocysteine (AdoHcy) hydrolase. In this study, we describe the metabolic fate of the 2-methylpropyl ester of AHPA (AHPA-iBu) in murine L929 cells. When AHPA-iBu was included in the culture medium, it was taken up rapidly by murine L929 cells. The uptake was time- and concentration-dependent, resulting in the intracellular accumulation of the free acid, AHPA. Treatment with AHPA-iBu caused inhibition of cellular AdoHcy hydrolase in both a time- and a dose-dependent manner. Complete inhibition of the enzyme was achieved after a 1-hr incubation in culture medium containing 50 μM AHPA-iBu. The inhibition of the enzyme caused cellular accumulation of AdoHcy and a significant increase in the ratio of AdoHcy/S-adenosyl-l-methionine (AdoMet). Partial recovery of the AdoHcy hydrolase activity in L929 cells treated with 50 μM AHPA-iBu was observed after 24 hr. This recovery of enzyme activity was paralleled by a significant decrease in the cellular levels of AdoHcy and the ratio ofAdoHcy/AdoMet. AHPA-iBu also exerted an inhibition (ic50 = 0.17 μM) of vaccinia virus plaque formation in monolayers of L929 cells. A 1 μm concentration of AHPA-iBu, which caused 80% inhibition of plaque formation, produced a 17-fold increase in AdoHcy content in drug-treated, virus-infected cells versus non-drug-treated, virus-infected cells and a 15% undermethylation of the poly(A)+ RNA. These data show that AHPA-iBu is a prodrug for AHPA which inhibits cellular AdoHcy hydrolase. The inhibition of this enzyme elevates cellular levels of AdoHcy, creating an unfavorable environment which suppresses replication of vaccinia virus. © 1990.