In this study the theoretical Gaussian-2 K+/Na+ binding affinities (enthalpies) at 0 K (in kJ mol−1) for six amides in the order: formamide (109.2/138.5) < N-methylformamide (117.7/148.6) < acetamide (118.7/149.5) < N,N-dimethylformamide (123.9/156.4) < N-methylacetamide (125.6/157.7) < N,N-dimethylacetamide (129.2/162.6), reported previously (Siu et al., J. Chem. Phys. 2001; 114: 7045–7051), were validated experimentally by mass spectrometric kinetic method measurements. By monitoring the collision-induced dissociation (CID) of K+/Na+-bound heterodimers of the amides, the relative affinities were shown to be accurate to within ±2 kJ mol−1. With these six theoretical K+/Na+ binding affinities as reference values, the absolute K+/Na+ affinities of imidazole, 1-methylimidazole, pyridazine and 1,2-dimethoxyethane were determined by the extended kinetic method, and found to be consistent (to within ±9 kJ mol−1) with literature experimental values obtained by threshold-CID, equilibrium high-pressure mass spectrometry, and Fourier transform ion cyclotron resonance/ligand-exchange equilibrium methods. A self-consistent resolution is proposed for the inconsistencies in the relative order of K+/Na+ affinities of amides reported in the literature. These two sets of validated K+ and Na+ affinity values are useful as reference values in kinetic method measurements of K+/Na+ affinity of model biological ligands, such as the K+ affinities of aliphatic amino acids. Copyright © 2004 John Wiley & Sons, Ltd.