The interaction of benzyl isothiocyanate (in concentrations of 2·5–250 mg benzyl‐ITC g−1 protein) with myoglobin leads to the formation of derivatives which have been characterised in terms of their solubility, free epsilon amino groups, content of tryptophan and its quenching, as well as their molecular properties determined with electrophoretic and chromatographic methods. The reaction takes place primarily at the epsilon amino groups of lysine, whose content decreases depending on the concentration of the benzyl‐ITC present. A second possibility is the reaction of the secondary amino group present in tryptophan, whose concentration also decreases. These reactions increased the electrophoretical mobility during PAGE in the presence of urea, as a result of which the isoelectric points shifted from 7·33–7·45 to 5·13–5·6 as shown by means of the isoelectrical focusing technique. The molecule size (size exclusion chromatography) decreased as a result of increased hydrophobicity (RP‐HPLC). A slight polymerisation with an increase in the content of dimer (38 kDa, SDS‐PAGE) was observed. The derivative with the highest degree of derivatisation (250 mg benzyl‐ITC g−1 protein) showed an unexpected behaviour with all its investigated properties, which can be explained as a result of structural changes taking place during the process of derivatisation.