Lipopolysaccharide (LPS) was isolated from Thiobacillus ferrooxidans IFO 14262 by the hot phenol-water extraction procedure. The O-specific polysaccharide, liberated from LPS by mild acetic acid hydrolysis, had a branched pentasaccharide repeating-unit composed of d-glucose, l-rhamnose, d-rhamnose, and 3- O-methyl- l-rhamnose in approximate molar ratios of 2:1:1:1. On the basis of methylation analysis, 1H and 13C NMR spectroscopy, including 2D shift-correlated (COSY) and 1D NOE spectroscopy, the structure for the repeating unit of the O-specific polysaccharide was established, and the assumed biological repeating unit indicated.