To refine our understanding of the mutational spectra one might expect on exposure of human cells to nitric oxide (NO), we have treated the plasmid pSP189 at pH 7.4 with two compounds that generate NO spontaneously in solution, and then sequenced the mutations found when the treated plasmid was transfected into human Ad293 cells and allowed to replicate. G.C-->A.T transitions were the most abundant mutation observed with these NO donor drugs, whereas in previous work, A.T-->G.C transitions predominated when nitric oxide gas was bubbled through the plasmid solution under otherwise identical conditions. A difference in reactive intermediates formed in solution- versus gas-phase NO exposure was demonstrated by treating buffered 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) or ferrocyanide, in the presence or absence of azide, aerobically with preformed solutions of NO, with solutions of the two NO-releasing compounds, or with gaseous mixtures of equimolar NO/O2 in air; oxidation of these substrates was extensive with the gas-phase NO source whether azide was present or not, while azide almost completely quenched the oxidation pathway in the solution-phase reactions.