Each of the following thiols react with a range of alkyl nitrites in water at 25 °C in the pH range 6–13 to give the corresponding thionitrites in solution: L-cysteine, N-acetyl-L-cysteine, L-cysteine methyl ester, L-cysteine ethyl ester, glutathione and thioglycolic acid. The pH-dependence of the rate constant clearly shows that reaction occurs only via the thiolate anions RS–. For N-acetyl-L-cysteine and thioglycolic acid only one RS– species is possible and a quantitative kinetic analysis yields pKa values for RSH ionisation in good agreement with the literature values. For each of the remaining thiols two thiolate ions (NH2RS– and NH3RS–) are possible and the measured rate constants for all alkyl nitrites generally followed the total thiolate ion concentration (as a function of pH) obtained from the published microscopic pKa values. An alternative approach of fitting the kinetic results to the concentration curve by computer yields microscopic pKa values generally in good agreement with the literature values. One exception is L-cysteine where the measured (pKa)D value (for NH2-RSH → NH2RS–) differs significantly from the literature value. With simple alkyl nitrites (ethyl, isopentyl, isopropyl and t-butyl) steric effects appear to be the major influences in reactivity, whereas electron-withdrawing substituents in the 2-position greatly increase the rate constants. The reactions of 2,2,2-trichloroethyl nitrite were too fast to measure at all pH values, whereas the reaction of 2,2-dichloroethyl nitrite could only be followed kinetically in the pH range 6–8.25, even by stopped-flow spectrophotometry. The possible relevance of these reactions to the vasodilatory action of alkyl nitrites is discussed. The kinetic results for thiolate nitrosation by N-methyl-N-nitrosotoluene-p-sulphonamide also correlate well with the total concentration of the thiolate ion, with the interesting exception of the reactions of the carboxylic esters of L-cysteine, where there appears to be a preference for reaction via the NH2RS– form.