The kinetic resolution of a range of methyl-substituted 1-oxaspiro[2.5]octanes by yeast epoxide hydrolase (YEH) from Rhodotorula glutinis has been investigated. The structural determinants of substrate specificity and stereoselectivity of YEH toward these substrates appeared to be the configuration of the epoxide ring and the substitution pattern of the cyclohexane ring. For all compounds tested, O-axial epoxides were hydrolyzed faster than the corresponding O-equatorial compounds. In concern of the ring substituents, YEH preferred methyl groups on the Re side of the ring. Placement of substituents close to the spiroepoxide carbon decreased the reaction rate but increased enantioselectivity. YEH-catalyzed kinetic resolutions of 4-methyl 1-oxaspiro[2.5]octane epimers were most enantioselective (E > 100).