The pathway of L-sorbose metabolism in Agrobacterium tumefaciens strain B6 was determined to be: L-sorbose leads to D-glucitol (sorbitol) leads to D-fructose leads to D-fructose-6-phosphate leads to D-glucose-6-phosphate. The reduction of L-sorbose and the oxidation of D-glucitol were mediated by NADPH- and NAD+-linked oxidoreductases, respectively. The intermediates, D-glucitol and D-fructose, were isolated from in vitro reaction mixtures by column chromatography on Dowex 1-borate, and identified enzymatically. D-Fructose was identified chemically by its 1H-NMR spectrum and the IR spectrum and the melting point of the fructosazone. D-Glucitol was characterized chemically by the melting point and the IR spectrum of its hexaacetate. A. tumefaciens ICPB TT111, a representative of another genetic race of Agrobacterium, lacked L-sorbose reductase and therefore failed to grow on L-sorbose; it grew normally on D-glucitol.