De-O-acylated lipopolysaccharides (LPS) of three polymyxin-resistant Salmonella typhimurium pmrA mutants and their parent strains were analysed by 31P-NMR (nuclear magnetic resonance) in order to assess, in relation to polymyxin resistance, the types and degree of substitution of phosphates of the LPS and lipid A. In the pmrA mutant LPS phosphate diesters predominated over phosphate monoesters, whereas the latter were more abundant in the parent wild-type LPS. The increase in the proportion of phosphate diesters was traced to both the core oligosaccharide and the lipid A part. In the latter, the ester-linked phosphate at position 4' was to a large extent (79-88%) substituted with 4-amino-4-deoxy-L-arabinose, whereas in the wild-type LPS the 4'-phosphate was mainly present as monoester. In each LPS, regardless of the pmrA mutation, the glycosidically linked phosphate of lipid A was largely unsubstituted.