Two microorganisms showing high omega-transaminase activity (Klebsiella pneumoniae JS2F and Bacillus thuringiensis JS64) were screened by the enrichment method using (S)-alpha-methylbenzylamine (alpha-MBA) as a sole nitrogen source. Optimal carbon and nitrogen sources for enzyme induction and the properties of omega-transaminases were investigated. omega-Transaminase from B. thuringiensis JS64 was highly enantioselective (E = 75.3) for (S)-enantiomer of alpha-MBA and showed remarkable stability. However, omega-transaminase showed severe product inhibition by acetophenone. An aqueous/organic two-phase system was introduced to overcome this problem. Through solvent screening, cyclohexanone and ethyl acetate were selected as the best organic phases. The acetophenone-extracting capacity of the solvent and the biocompatibility of the solvent to the cell were important determinants in the reaction rate at high concentrations of alpha-MBA. The reaction rate of omega-transamination was strongly influenced by the volume ratio of organic phase to aqueous phase as well as agitation speed in the biphasic mixture. Using the optimal volume ratio (V-org:V-aq = 1:4) in the biphasic system with cyclohexanone, the reaction rate of omega-transaminase under vigorous mixing conditions increased ninefold compared with that in the monophasic aqueous system. At the same optimal conditions, using whole cells, 500 mM alpha-MBA could be resolved successfully to above 95% enantiomeric excess of (R)-alpha-MBA with ca. 51% conversion.