The object of the present study is to investigate whether the physiologically dominant thiol compounds such as GSH and cysteine or their nitrosothiol compounds affect the formation of the iron- N -methyl-D-glucamine dithiocarbamate [(MGD)(2)Fe(2+)]-nitric oxide complex. The present study provided experimental evidence that physiological concentrations of GSH (approx. 5 mM) and L-cysteine (approx. 0.5 mM) accelerated the formation of the (MGD)(2)Fe(2+)-NO complex from nitrite by two and three times respectively. The rate constants for the reduction of (MGD)(3)Fe(3+) to (MGD)(2)Fe(2+) by GSH and cysteine were calculated as 1.3 and 2.0x10(2) M(-1).s(-1) respectively. Furthermore, depletion of GSH was demonstrated in PC12 cells, and thiol compounds enhanced the formation of reactive oxygen species by the (MGD)(2)Fe(2+) complex by accelerating its redox turnover. The main effect of the physiological concentration of thiols was the reduction of (MGD)(3)Fe(3+). S -nitrosoglutathione spontaneously reacted with (MGD)(2)Fe(2+) to produce the (MGD)(2)Fe(2+)-NO complex with a 1:2 stoichiometry. In fact, (MGD)(2)Fe(2+) was as good an indicator of nitrosothiols as it was of NO itself. The present study elucidates the difficulties of utilizing the (MGD)(2)Fe(2+) complex for the quantification of NO in biological samples, especially in vivo.