The nitrosation of various thiols and morpholine by oxygenated (NO)-N-. solutions at physiological pH was investigated. The formation rates and the yields of the nitroso compounds were determined using the stopped-flow technique. The stoichiometry of this process has been determined, and is given by 4(.)NO + O-2 + 2RSH/2RR’NH --> 2RSNO/2RR’NNO + 2NO(2)(-) + 2H(+). Kinetic studies show that the rate law is -d[O-2]/dt k(l)[(NO)-N-.](2)[O-2] with k(l) = (2.54 +/- 0.26) x 10(6) M(-2) s(-1) and -d[(NO)-N-.]/dt = 4k(l)[(NO)-N-.](2)[O-2] with 4k(l) = (1.17 +/- 0.12) x 10(7) M(-2) s(-1), independent of the kind of substrate present. The kinetic results are identical to those obtained for the autoxidation of (NO)-N-., indicating that the rate of the autoxidation of (NO)-N-. is unaffected by the presence of thiols and amines. The nitrosation by (NO)-N-. takes place only in the presence of oxygen, and therefore the rate of the formation of S-nitrosothiols from thiols and oxygenated (NO)-N-. solution is relatively slow in biological systems. Under physiological conditions where [(NO)-N-.] < 1 mu M and [O-2] < 200 mu M, the half-life of the nitrosation process exceeds 7 min. Therefore, this is an unlikely biosynthetic pathway for the formation of S-nitrosothiols. As such, S-nitrosothiols cannot serve as carrier molecules of (NO)-N-. in vivo. The rate-determining step of the nitrosation of thiols and amines by oxygenated (NO)-N-. solution is the formation of ONOONO (or ONONO2 or O2NNO2), which is the precursor of (NO2)-N-. and N2O3 The stoichiometry of the nitrosation process suggests that (NO2)-N-. and/or N2O3 are the reactive species. We have demonstrated that (NO2)-N-. initiates the nitrosation process unless it is scavenged faster by (NO)-N-. to form N2O3. The latter entity is also capable of directly nitrosating thiols and amines with rate constants exceeding 6 x 10(7) M(-1) s(-1).