An O-haptenic material (LI-fraction) was isolated from cytoplasmic and cell-wall preparations of the rough mutant Salmonella typhimurium 395 MR10. The yield from the cell-wall preparation, virtually free from cytoplasmic contamination, was approximately ten times higher than that from the cytoplasmic preparation. The LI-fraction obtained from cell-walls was less contaminated with extraneous polysaccharide material. After purification of the LI-fraction with ribonuclease treatment and Sephadex G-100 gel chromatography structural studies using methylation analysis could be performed. The hapten showed the same structural features as the O side-chains of the parent strain S. typhimurium 395 MS. The average number of repeating units in the hapten was 23 as compared to 11 in the O side-chain of the parent strain. Molecular weight determination with Sepharose 6 B gel chromatography revealed that the LI-fraction was polydisperse with a weight average of 26,300 and a number average of 21,800.