Applications of cysteine-insertion and thioether linkage approaches to the preparation of a number of bioactive peptide conjugates are reported. Peptides containing epitopes from (i) herpes simplex virus type 1 glycoprotein D, (ii) a specific N-terminal β-amyloid epitope recognized by therapeutically active antibodies, and (iii) a GnRH-III peptide from sea lamprey with antitumour activity, were elongated with Cys residues and attached to a chloroacetylated tetratuftsin derivative carrier via a thioether linkage either directly, or by insertion of a spacer. The structures and molecular homogeneity of all the peptide conjugates were ascertained by HPLC, MALDI and electrospray mass spectrometry. The use of a spacer such as an oligoglycine or GFLG-tetrapeptide gave an increased yield in the conjugation reaction and enhanced reaction rates. In the formation of cysteinyl-thioether linkages, it was found that the position of flanking Cys residues markedly influenced the conjugation reaction and the formation of intermolecular epitope disulfide-dimers. C-terminal Cys residues gave thioether conjugates with significantly diminished epitope-dimerization, while Cys at the N-terminal caused rapid disulfide-dimerization, thereby preventing efficient conjugation. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.