The nitric oxide (N = O) free radical exhibits potent cytocidal, mutagenic and vasodilatory properties. We have examined the hypothesis that the hydroxynitrosamino functionality (see sequence in text), which occurs naturally in antineoplastic and antihypertensive agents, will directly generate N = O following peroxidatic 1-electron oxidation. Cupferron (see sequence in text) is indeed an excellent (k greater than 10(7) m-1 s-1) substrate for horseradish peroxidase. The products are N = O and nitrosobenzene (phi - N = O) which are generated and consumed as follows. First, cupferron is oxidized by the classical peroxidatic mechanism to form an unstable nitroxide free radical (see sequence in text) which then forms N = O and phi - N = O spontaneously (see sequence in text). The N = O then reacts with phi - N = O to reform cupferron (see sequence in text) or with the enzyme to generate the characteristic peroxidase--N = O chromophore. Simultaneously, in a competitive reaction with O2, the N = O is converted to NO-2 (4N = O + O2 + 2H2O------------4NO-2 + 4H+). The reactivity of hydroxynitrosamino compounds with horseradish peroxidase is in the order cupferron greater than hydroxynitrosaminomethane greater than alanosine. These model reactions, involving direct oxidation of the hydroxynitrosamino moiety, comprise a novel pathway for the biological production of N = O.