The distribution of 3-ketolactose formation among members of the alpha subclass of the Proteobacteria was investigated by the Benedict reagent test and by a more sensitive quantitative method in which high-performance liquid chromatography was used. 3-Ketolactose formation activity was found in strains of Agrobacterium biovar 1 and in strains of eight species of the genus Sphingomonas, including Sphingomonas paucimobilis (the type species), which belong to the alpha-2 and alpha-4 subclasses of the Proteobacteria, respectively. Weak activity was found in Erythrobacter longus IFO 14126T(T = type strain), a member of the alpha-4 subclass of the Proteobacteria. The ketosugar was not produced by members of the other taxa of the alpha subclass of the Proteobacteria tested. The ketosugar was isolated from culture broths of S. paucimobilis IFO 13935Tand Sphingomonas yanoikuyae IFO 15102Tand was identified as 3-ketolactose [4-O-(β-D-xylo-hexopyranosyl-3-ulose)-D-glucopyranose] by chromatographic analyses and 1H nuclear magnetic resonance spectroscopy.