New approaches to electrospray ionization mass spectrometry (ESI-MS) -with exact compositional assignments - of small (Au-25) nanoparticles with uniform and mixed protecting organothiolate monolayers are described. The results expand the scope of analysis and reveal a rich chemistry of ionization behavior. ESI-MS of solutions of phenylethanethiolate monolayer-protected gold clusters (MPCs), Au-25(SC2Ph)(18), containing alkali metal acetate salts (MOAc) produce spectra in which, for Na+, K+, Rb+, and Cs+ acetates, the dominant species are MAu25(SC2Ph)(18)(2+) and M2Au25(SC2Ph)(18)(2+). Li+ acetates caused ligand loss. This method was extended to the analysis of Au-25 MPCs with mixed monolayers, where thiophenolate ( - SPh), hexanethiolate ( - SC6), or biotinylated ( - S - PEG - biotin) ligands had been introduced by ligand exchange. In negative-mode ESI-MS, no added reagents were needed in order to observe Au-25(SC2Ph)(18)(-) and to analyze mixed monolayer Au-25 MPCs prepared by ligand exchange with 4-mercaptobenzoic acid, HSPhCOOH, which gave spectra through deprotonation of the carboxylic acids. Adducts of tetraoctylammonium (Oct(4)N(+)) with -SPhCOO- sites were also observed. Mass spectrometry is the only method that has demonstrated capacity for measuring the exact distribution of ligand-exchange products. The possible origins of the different Au-25 core charges (1-, 0, 1+, 2+) observed during electrospray ionization are discussed.