Cyclohexanone monooxygenase (E.C. 1.14.13.22) from Acinetobacter sp. NCIB 9871 has been expressed in baker's yeast (Saccharomyces cerevisiae) to create a general reagent for asymmetric Baeyer-Villiger oxidations. This "designer yeast" approach combines the advantages of using purified enzymes (single catalytic species, no overmetabolism, etc.) with the benefits of whole-cell reactions (experimentally simple, no cofactor regeneration necessary, etc.). The yeast reagent was used to systematically examine a series of 2-, 3-, and 4-substituted cyclohexanones (R = Me, Et, n-Pr, i-Pr, allyl, n-Bu), almost all of which were oxidized to the corresponding epsilon-caprolactones in good yields and high enantioselectivities (typically greater than or equal to 95%). Mesomeric 4-substituted cyclohexanones were oxidized to epsilon-caprolactones in greater than or equal to 92% ee. The engineered yeast strain also effected kinetic resolutions of 2-substituted cyclohexanones with enantioselectivity values greater than or equal to 200 for substituents larger than methyl. The behavior of 3-substituted cyclohexanones depended upon the size of the substituent. The engineered yeast strain cleanly converted the antipodes of 3-methyl- and 3-ethylcyclohexanone to divergent regioisomers. On the other hand, for cyclohexanones with larger substituents (n-Pr, allyl, n-Bu), both antipodes were oxidized by the enzyme to a single regioisomer. In these cases, the observed enantioselectivities were due to a combination of a modest preference for one enantiomer by the enzyme and an unfavorable conformational preequilibrium required prior to binding of the less-favored antipode, a phenomenon we refer to as substrate-assisted enantioselectivity.