Biocatalysed synthesis of nucleosides was carried out using immobilised whole cells of Escherichia coli ATCC 47092, Enterobacter gergoviae CECT 857 and Citrobacter amalonaticus CECT 863. The synthesis of adenosine from uridine was used as reaction model to test the biocatalysts. Reactions were carried out using non-growing cells. Maximum activity was obtained with cells harvested at the beginning of the stationary phase. Immobilization by whole cell entrapment was employed using different matrix such as alginate, agar, agarose and polyacrylamide. The percentage of monomer, the shaking speed, the catalyst load and nature of the matrix were optimized. In the first reutilization cycle, similar yields (80-95%) were obtained with both free and immobilized cells in the reaction model, although in the last case, longer reaction times were necessary. The immobilized cells can be reused at least for more than 30 times without significant loss of the catalytic activity. The immobilized biocatalysts have been used in the synthesis of different nucleosides. © 2004 Elsevier B.V. All rights reserved.