A biological conversion of L-sorbose from the mixture of glucose and fructose was studied. D-Sorbitol was produced efficiently from fructose by permeabilized Zymomonas mobilis cells and then converted to L-sorbose by Gluconobacter suboxydans. The process for the production of L-sorbose from D-sorbitol by the resting cells of G. suboxydans was also optimized with respect to the substrate concentration, oxygen tension and cell density of the reaction medium. Gluconate produced from glucose by Z. mobilis seemed to act as an inhibitor in the conversion reaction of D-sorbitol into L-sorbose by G. suboxydans. A two-stage fed-batch process, therefore, was developed to minimize the inhibitory effect of gluconate on the conversion reaction.