Three bacterial strains, A, B and C, were isolated from activated sludge as 2-sulfonato-fatty-acid-methyl-ester (alpha-SFMe)-degrading microorganisms. From the results of morphological, physiological and biochemical studies, and analyses of 16S rRNA gene sequences, isolate A was identified as Agrobacterium tumefaciens while B and C were Pseudomonas putida, respectively. To demonstrate their capability for the ultimate biodegradation of alpha-SFMe, the degradation kinetics have been investigated using C-14-alpha-SFMe and 2-C-14-labeled C-16-alpha-SFMe. The biodegradation was determined by measuring dissolved organic carbon (DOC) and released SO42-, in the shake-culture test, and evolved (CO2)-C-14 in the modified Organisation for Economic Go-operation and Development (OECD) test. In the shake culture test with C-14-alpha-SFMe, DOC removal was progressive throughout the test, Liberation of inorganic sulfate started after DOC removal and then rapidly increased. During the (CO2)-C-14 evolution tests, the mineralization of radiolabeled carbon started quickly and reached about 80% of the initially added radioactivity at the end of the tests. The results obtained indicated that all of the isolates had the capability for ultimately degrading alpha-SFMe through the oxidation of the alkyl carbons and desulfonation (cleavage of the C-S linkage).