Bacillus coagulans CECT 12, Bacillus psychrosaccharolyticus CECT 4074, Lactobacillus sp. CECT 4219 and Psychrobacter immobilis CECT 4492 have been characterised, after a taxonomic screening, as nucleoside 2′-deoxyribosyltransferase (type II) (E.C. 2.4.2.6) producers. They are selective for 6-aminopurine compared to 6-oxopurines and for 2′-deoxyuridine compared to thymidine. The synthesis of 2′-deoxyribosylnucleosides is performed in Tris/HCl at T = 57 °C, lower than the 70 °C commonly used in the literature. These microorganisms do not produce hypoxanthine by decomposition of adenine nucleoside as described for other strains. 2′-Deoxyribosylnucleosides of 5-chloro- or 5-fluoruracil can be obtained in a one-pot reaction. The strains can be immobilized in calcium alginate or calcium pectate producing biocatalysts active and selective in the synthesis of 2′-deoxyadenosine at 57 °C with high yields and without secondary reactions. In the present study two psychrotrophic microorganisms are described for the first time, B. psychrosaccharolyticus and P. immobilis, which show high nucleoside 2′-deoxyribosyltransferase activity both in free conditions and immobilized in calcium pectate. © 2006 Elsevier Inc. All rights reserved.