Sorbitol dehydrogenase (SDH, EC 1.1.1.14) was purified to homogeneity from a cell-free extract of Pseudomonas sp. KS-E1806 by chromatographies with QAE-Sephadex A-50, QAE-Toyopearl 550C, and Bio-Gel A-1.5m. The molecular weight of the enzyme was estimated to be 64,500 and 27,400 by gel filtration and SDS-PAGE, respectively, suggesting that it has a dimeric structure. The enzyme was stable from pH 5.5 to 10.5, and below 40 degrees C for 30 min at pH 9.0. D-Sorbitol and galactitol were good substrates of the enzyme, whereas it acted only slightly on xylitol and D-mannitol. Tbe enzyme was superior to sheep liver SDH with respect to substrate specificity and pH stability, and it was more thermostable than a previously reported Pseudomonas sp, SDH. Compared with other known SDHs, the enzyme from Pseudomonas sp. KS-E1806 has several advantages for practical use in the enzymatic analysis of D-sorbitol.