Electrospray ionization mass spectrometry (ESI-MS) was used to study the binding interactions of two series of ruthenium complexes, [Ru(phen)(2)L](2+) and [RuL2'(dpqC)](2+), to a double stranded DNA hexadecamer, and derive orders of relative binding affinity. These were shown to be in good agreement with orders of relative binding affinity derived from absorption and circular dichroism (CD) spectroscopic examination of the same systems and from DNA melting curves. However, the extent of luminescence enhancement caused by the addition of DNA to solutions of the ruthenium complexes showed little correlation with orders of binding affinity derived from ESI-MS or any of the other techniques. Overall the results provide support for the validity of using ESI-MS to investigate noncovalent interactions between metal complexes and DNA.