Low-molecular-mass S-nitroso compounds (R-S-N O) are potent vasodilators and inhibitors of platelet aggregation. This work describes the electrospray ionization mass spectrometric (ESI-MS) analysis of physiological and synthetic low-molecular-mass S-nitroso compounds and their thiols including S-nitrosoglutathione, S-nitrosocysteine, glutathione and cysteine. Mass spectra of the unlabeled and S- 1 5 N-labeled low-molecular-mass S-nitroso compounds investigated are characterized by abundant cations due to [M+H] + , [M+Na] + , [(M+H)-NO] + , [2 M+H] + , and [(2 M+H)-2NO] + . Mass spectra of low-molecular-mass thiols are characterized by abundant cations due to [M+H] + , [M+Na] + and [2M+H] + . Using off-line electrospray ionization tandem mass spectrometry we unequivocally identified S-[ 1 5 N]nitrosoglutathione in human red blood cells formed after their incubation with S-[ 1 5 N]nitrosocysteine. These results suggest that ESI-MS in combination with an appropriate liquid chromatographic system should be a useful analytical approach for the on-line quantitative determination of low-molecular-mass S-nitroso compounds in biological fluids in the presence of their thiols and nitrite. Considerations were made about on-line ESI-MS and quantitative measurements.