The initial ionization reaction in matrix-assisted laser desorption/ionization (MALDI) was examined on the basis of the appearance of photoelectrons. The threshold laser fluence for the ejection of photoelectrons from 2,5-dihydroxybenzoic acid (DHB), sinapinic acid (SA), and trihydroxyacetopheone (THAP) on stainless steel (SS) substrates was 0.05, 0.41, and 8.39 mJ/cm(2), respectively. These values are considerably lower than those for MALIN ions, indicating that the electron detachment likely precedes other ionization reactions. The SS substrate played an insignificant role in the production of photoelectrons because suspended DHB produced a photoelectron signal similar to DHB on the SS surface, and decreasing the DHB thickness on the SS reduced the photoelectron intensity. For crystalline DHB and SA, the photoelectron intensity increased with the laser (337 nm) fluence in a relationship of less than second order, suggesting considerable reductions of ionization potentials in comparison with free molecules. According to ab initio calculations, the ionization potential of DHB clusters reduces as the cluster size increases from monomer to octamer. The impact of these abundant electrons on the ion production in MALDI is discussed.