Selective biocatalyzed synthesis of 2′-deoxyadenosine from 2′-deoxypyrimidine nucleosides was carried out using free or immobilized whole cells. The reaction was performed at 57°C without secondary reactions. Two psychrotrophic microorganisms, Bacillus psychrosaccharolyticus and Psychrobacter immobilis, are described for the first time as active and specific strains for the synthesis of 2′-deoxyadenosine. Adenosine deaminase activity was not detected. Whole cells were immobilized in different matrixes. Calcium alginate and calcium pectate gave the best biocatalysts. The synthesis of 2'-deoxyadenosine follows an apparent first order kinetic expression. External mass transfer control was negligible as deduced from ks, NA, and Ω values. Internal mass transfer was the rate controlling step according to ηT and φ values. © 2007 Wiley Periodicals, Inc.