Factors governing the molecular ion yields of amino acids and peptides have been studied using fast atom bombardment (FAB) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) in positive-ion mode. The ion yields of protonated amino acids under FAB conditions are dependent on proton affinity (PA), hydrophobicity, and aromaticity of amino acids. Both PA and hydrophobicity contribute to an increase in the ion yields, while aromaticity contributes to a decrease. In MALDI, the ion yields increase linearly with the increase of PA of amino acids with the exception of lysine. In both FAB and MALDI experiments with peptides, the presence of arginine residues is essential for producing abundant protonated peptides. In FAB, the presence of aliphatic and hydrophobic amino acids (leucine and isoleucine) increases the ion yields of protonated peptides, while some hydrophilic amino acids (aspartic acid and asparagines) decrease the ion yields. The presence of two or more arginine residues does not give higher ion yields in FAB. In MALDI, the presence of aromatic amino acids (phenylalanine and tyrosine) enhances the signals for protonated peptides. Thus, physicochemical factors of individual amino acids cooperatively affect the ion yields of protonated amino acids and peptides. These factors governing the ion yields in FAB and MALDI affect two processes, desorption and ionization, that can be considered independently. Copyright © 2005 John Wiley & Sons, Ltd.