Oligonucleotide‐directed mutagenesis of ctxB was used to produce mutants of cholera toxin B subunit (CT‐B) altered at residues Cys‐9, Gly‐33, Lys‐34, Arg‐35, Cys‐86 and Trp‐88. Mutants were identified phenotypically by radial passive immune haemolysis assays and genotypically by colony hybridization with specific oligonucleotide probes. Mutant CT‐B poly‐peptides were characterized for immunoreactivity, binding to ganglioside GM1, ability to associate with the A subunit, ability to form holotoxin, and biological activity. Amino acid substitutions that caused decreased binding of mutant CT‐B to ganglioside GM1 and abolished toxicity included negatively charged or large hydrophobic residues for Gly‐33 and negatively or positively charged residues for Trp‐88. Substitution of lysine or arginine for Gly‐33 did not affect immunoreactivity or GM1 ‐binding activity of CT‐B but abolished or reduced toxicity of the mutant holotoxins, respectively. Substitutions of Glu or Asp for Arg‐35 interfered with formation of holotoxin, but none of the observed substitutions for Lys‐34 or Arg‐35 affected binding of CT‐B to GM1. The Cys‐9, Cys‐86 and Trp‐88 residues were important for establishing or maintaining the native conformation of CT‐B or protecting the CT‐B polypeptide from rapid degradation in vivo.