The kinetic resolution of a series of acetates of tertiary alcohols bearing a nitrogen substituent has been studied by using several recombinant carboxyl esterases and variants thereof expressed in E. coli. Most of the enzymes were active in the conversion of these tertiary alcohols and excellent enantioselectivities were achieved in the synthesis of three 2-(pyridin-x-yl)but-3-yn-2-ols with the nitrogen atom in the pyridine ring in the 2′-, 3′-, and 4′-positions. The resolution of tertiary cyanohydrins proved to be more difficult as the enantioselectivity of the enzymes was generally lower. Nevertheless, (S)-1-cyano-2,2,2-trifluoro-1- phenylethyl acetate was obtained with 99% ee. The results show that the limited substrate range of the individual enzymes in the synthesis of a series of tertiary alcohols can be efficiently overcome by using a combination of different enzymes. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.