The tert-butylation of the tryptophan indole ring during the removal of tert-butyloxycarbonyl groups under different conditions was studied. The incorporation of tert-butyl groups was measured by a 1H-NMR technique. It was established, that the rate and specificity of the tert-butylation is highly dependent on the character of the cleaving reagents used and on the environment of the tryptophan residue in the peptide chain.