Dioxolane nucleosides, in which an oxygen replaces the carbon in the 3' position in the ribose moiety of 2',3'-dideoxy nucleosides, are powerful antiviral and anticancer drugs. However, their synthesis remains challenging since it must control the relative and absolute stereochemistry of two stereocenters in the five-membered ring. Several promising routes yield a key intermediate dioxolane as a mixture of diastereomers (epimers at the 2- position of the dioxolane), but separation of these diastereomers by silica gel chromatography is tedious and expensive. In this paper, we report that two inexpensive, commercially available proteases-α-chymotrypsin and bovine pancreatic protease-discriminate between the cis and trans diastereomers of 2(R,S)-benzyloxymethyl-1,3-dioxolane-4(S)-carboxylic acid methyl ester. Although hydrolysis occurs at a carboxyl group three bonds away from the 2- stereocenter, the diastereoselectivity is high (D = 29-35, favoring trans). We discovered these selective hydrolases by screening a library of 91 commercial hydrolases with our previously developed stereoselectivity screens that use pH indicators. A small-scale α-chymotrypsin-catalyzed hydrolysis of a 2:1 mixture of cis- and trans-dioxolane methyl esters yielded the desired cis-dioxolane methyl ester in > 98% diastereomeric excess and 55% overall yield (67% was the maximum possible yield). Computer modeling of transition- state analogues of both diastereomers in the active site of α-chymotrypsin suggests that stereoselectivity arises because the slow-reacting diastereomer binds in a nonproductive orientation.