Positive ion electrospray ionization mass spectra of polypeptides are usually obtained from solutions that are acidified and therefore contain relatively high concentrations of anions. The present study describes an investigation of the effects of these ubiquitous anions on the positive ion electrospray ionization mass spectra of peptides and proteins. Certain anionic species in the spray solutions were observed to cause a marked decrease in the net average charge of peptide and protein ions in the mass spectra compared to the average charge measured in the absence of these anions. This charge neutralization effect was found to depend solely on the nature of the anionic species and was independent of the source of the anion (acid or salt), with the propensity for neutralization following the order: CCl3COO->> CF3COO->> CH3COO- approximately Cl-. A mechanism for the observed charge reduction effect is proposed that involves two steps. The first step occurs in solution, where an anion pairs with a positively charged basic group on the peptide. The second step occurs during the process of desolvation or in the gas phase, where the ion pair dissociates to yield the neutral acid and the peptide with reduced charge state. The different propensities for charge neutralization of the different anionic species is presumed to reflect the avidity of the anion-peptide interaction. These findings demonstrate that any attempt to correlate the distribution of charge states observed on proteins in the gas phase (by positive ion electrospray ionization mass spectrometry) with the net charge residing on the protein in solution will require that the described anion effect be taken into account. In addition, it appears that some control over the distribution of charge states on peptides and protein ions can be exercised by an appropriate choice of anion in the electrospray solution.