Atomically precise alloying and de-alloying processes for the formation of Ag–Au and Cu–Au nanoparticles of 25-metal-atom composition (referred to as AgxAu25−x(SR)18 and CuxAu25−x(SR)18, in which R=CH2CH2Ph) are reported. The identities of the particles were determined by matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS). Their structures were probed by fragmentation analysis in MALDI-MS and comparison with the icosahedral structure of the homogold Au25(SR)18 nanoparticles (an icosahedral Au13 core protected by a shell of Au12(SR)18). The Cu and Ag atoms were found to preferentially occupy the 13-atom icosahedral sites, instead of the exterior shell. The number of Ag atoms in AgxAu25−x(SR)18 (x=0–8) was dependent on the molar ratio of AgI/AuIII precursors in the synthesis, whereas the number of Cu atoms in CuxAu25−x(SR)18 (x=0–4) was independent of the molar ratio of CuII/AuIII precursors applied. Interestingly, the CuxAu25−x(SR)18 nanoparticles show a spontaneous de-alloying process over time, and the initially formed CuxAu25−x(SR)18 nanoparticles were converted to pure Au25(SR)18. This de-alloying process was not observed in the case of alloyed AgxAu25−x(SR)18 nanoparticles. This contrast can be attributed to the stability difference between CuxAu25−x(SR)18 and AgxAu25−x(SR)18 nanoparticles. These alloyed nanoparticles are promising candidates for applications such as catalysis.