1,5-Diaminonaphthalene (DAN) has been described as an interesting and effective matrix for matrix-assisted laser desorption/ionization (MALDI) experiments in positive ion mode, being able to activate in-source decomposition phenomena and, when employed for the analysis of proteins containing disulphide bridge(s), being able to activate reduction processes, resulting in disulphide bridge cleavage. The mechanisms of the DAN reactivity have been studied in detail, and the results indicate that the reduction properties of the matrix are of a radical nature. In the present study the structure of the reactive species produced by DAN, responsible for its reductive properties, has been investigated by accurate mass measurements and tandem mass spectrometry (MS/MS) experiments. Contrary to what is usually observed by laser irradiation of other MALDI matrices (with the sole formation of the MH+ ion of the matrix), DAN leads to the formation of odd-electron molecular ions M+•. This can be rationalized by the occurrence of two photon pooling processes, due to the low ionization energy of DAN. Thus the M+• ion of DAN can be considered responsible for both analyte protonation and disulphide bond reduction and some mechanisms are proposed for this behaviour. Copyright © 2011 John Wiley & Sons, Ltd.