An assay regimen for the detection of enantioselective esterase activity in bacteria and yeasts is described. This program was used to screen 167 culture collection strains and soil isolates for activity toward 2-substituted esters and yielded a Bacillus sphaericus strain that selectively hydrolyzes the R ester of ethyl mandelate, ethyl 2-hydroxypentanoate, and ethyl 2-hydroxyhexanoate. The latter substrate was resolved to the S ester in >99% enantiomeric excess after 56% hydrolysis.