Tyrosine is generally considered to be the physiological precursor of melanins and tyrosinase the enzyme responsible. However, recent studies have shown that also peroxidases are involved in the biosynthesis of melanins. These enzymes use hydrogen peroxide to oxidise various phenol substrates. In this paper, we used a substrate other than tyrosine, i.e. 5-hydroxytryptophan, to verify if its peroxidase/H2O2-mediated oxidation gave rise to the formation of melanin. We also subjected 5-hydroxytryptophan to the action of tyrosinase, for comparison purposes. We observed that both enzymes converted this substrate to melanin and that peroxidase, in the presence of hydrogen peroxide, was much more effective than tyrosinase in catalysing the oxidative polymerization of 5-hydroxytryptophan, with the formation of insoluble black melanin-like pigments. Samples deriving from the reaction-substrate enzyme were ultrafiltered at different times through an Amicon ultrafiltration cell equipped with an Amicon Diaflo XM-50 membrane, in order to remove the enzyme, and immediately lyophilised. The resulting samples were analysed by matrix assisted laser desorption/ionisation (MALDI) mass spectrometry, which clearly identified several oligomer species in the reaction mixture. This work was undertaken to investigate the possible precursors of neuromelanin and the enzyme responsible for melanogenesis in brain, since although the central nervous system does not contain tyrosinase, it is rich in peroxidase and hydrogen peroxide.