A better understanding of the origins of NO and HNO and their activities and biological functions requires accurate methods for their detection and quantification. The unique reaction of NO with nitronyl nitroxides such as 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (C-PTIO), which yields the corresponding imino nitroxides, is widely used for NO detection (mainly by electron paramagnetic resonance spectroscopy) and for modulation of NO-induced physiological functions. The present study demonstrates that HNO readily reacts with nitronyl nitroxides, leading to the formation of the respective imino nitroxides and hydroxylamines via a complex mechanism. Through the use of the HNO donor Angeli's salt (AS) with metmyoglobin as a competing agent, the rate constant for C-PTIO reduction by HNO has been determined to be (1.4 +/- 0.2) x 10(5) M(-1) s(-1) at pH 7.0. This reaction yields the corresponding nitronyl hydroxylamine C-PTIO-H and NO, which is trapped by C-PTIO to form (*)NO(2) and the corresponding imino nitroxide, C-PTI. (*)NO(2) oxidizes the nitronyl and imino nitroxides to their respective oxoammonium cations, which decay mainly via comproportionation with the nitronyl and imino hydroxylamines. When [AS] > [C-PTIO], the reduction of C-PTI by HNO proceeds, eventually converting C-PTIO to the corresponding imino hydroxylamine, C-PTI-H. Similar results were obtained for 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). It is concluded that nitronyl nitroxide is readily reduced by HNO to nitronyl hydroxylamine and is eventually converted into imino nitroxide and imino hydroxylamine. The yield of the imino hydroxylamine increases at the expense of the imino nitroxide as the ratio [AS](0)/[nitronyl nitroxide](0) is increased. Since the reaction of NO with nitronyl nitroxide yields only the corresponding imino nitroxide, nitronyl nitroxide can discriminate NO from HNO only when present at a concentration much lower than the total production of HNO.