The metabolism of 2,6-naphthalenedisulfonate (26NDS) by Pigmentiphaga sp. NDS-2 was analyzed by isolating a mutant (NDS2-002) which slowly grew on a minimal medium containing 26NDS as the sole source of carbon. Liquid chromatography/mass spectrometry (LC/MS) analysis of metabolic intermediates in this strain revealed that 5-sulfosalicylate (5SSA) is accumulated during the degradation of 26NDS. To analyze the lower metabolic pathway, a mutant strain NDS2-008, which could not grow either on 26NDS, 5SSA or gentisate, but could on succinate as the carbon source, was isolated. When the resting cells of NDS2-008 were incubated with 5SSA or gentisate, a substance deduced to be maleylpyruvate (Zhou et al., J. Bacteriol., 183, 700-708, 2001) was commonly detected upon HPLC analysis. These results suggest that Pigmentiphaga sp. NDS-2 degrades 26NDS via 5SSA, gentisate and maleylpyruvate as intermediates.