A freeze-dried whole-cell biocatalyst was prepared from Pseudomonas fluorescens and applied for the first time to regioselective synthesis of 1-beta-D-arabinofuranosylcytosine monoester in nonaqueous media. The catalytic performances of the bacterial cells were significantly enhanced by cultivation with a mixed carbon sources containing yeast extract and additional lipid-related substrates, especially the supplement of soybean oil. Cultivation of the cells supplemented with glucose, however, resulted in both low biomass and catalytic activities in the acylation. Taking into account the yield and 5'-regioselectivity of the cell-mediated reaction, yeast extract appeared to be the most suitable for cell cultivation, of all the tested nitrogen sources. Due to the fact that the nutrient concentrations and culture time are also crucial factors affecting the corresponding cell-catalyzed reaction, their effect on the catalytic performance of the cells were also investigated. The best soybean oil concentration, yeast extract concentration and culture time were 0.5% (w/v), 0.1% (w/v) and 48 h, respectively, under which the yield and 5'-regioselectivity of the reaction catalyzed by the cell biocatalyst reached 75.4% and 96.8%, respectively. Our results demonstrated that P.fluorescens whole cell was a green and economic alternative to enzymes for regioselective acylation of ara-C in non-aqueous media. (C) 2012 Elsevier Ltd. All rights reserved.