In the present study, we have investigated S-nitrosation of reactive thioredoxin (Trx) thiol groups in nitric oxide/superoxide system. We have found that Trx thiol groups are the targets for S-nitrosation by N2O3-like species generated in the system containing xanthine/xanthine oxidase (superoxide producing system) and DEA/NO-the *NO donating compound, however, they have shown low sensitivity to the *NO derived from DEA/NO. N2O3-dependent S-nitrosation of Trx at approximately 2-fold of NO excess compared to the superoxide amount resulted in dissociation and activation of apoptosis signal regulating kinase 1 (ASK1). However, approximately 4-fold of NO excess compared to a superoxide production preserved the level of dissociated ASK1 but decreased its activity due to the enzyme S-nitrosation.