The results of a screen for microbial esterases that have enantioselective activity for the hydrolysis of butyl 2-ethylhexanoate are described. The preliminary screen determined that a nocardio-form bacterial strain, NRRL 21057, exhibited significant activity in preferentially hydrolyzing the S enantiomer of butyl 2-ethylhexanoate. Molecular systematics methods identified NRRL 21057 as a strain of Nocardia corynebacteroides. A survey of phylogenetically related species in the genera Gordonia, Rhodococcus, and Nocardia strains demonstrated that N. corynebacteroides NRRL 21057 is the most active strain known for the specific hydrolysis of the R-isomer of butyl 2-ethylhexanoate and that it provides the S-isomer of 2-ethylhexanoate in 86% enantiomeric excess within 22 h.