Specific and nonspecific noncovalent dimer ions of oligonucleotides (ODNs) were observed when mixtures of complementary or noncomplementary strands were analyzed via negative ion electrospray ionization mass spectrometry. Dimer formation was concentration dependent and nearly always occurred when the concentration of ODN exceeded 100 µM. Dimers were observed even for short-length ODNs for which the melting temperature (T m) was well below the experimental temperature and which, therefore, would not be expected to form stable solution duplexes. The abundance of the heterodimer ions seems to correlate with the number of expected hydrogen bonds from Watson-Crick base pairing. As the energy of the incoming ion beam (orifice potential) was increased, the absolute and relative abundance of the dimer ions unexpectedly increased.