Cardiac sympathetic transmitter stores are reduced in the failing heart. In this study, we proposed to study whether the reduction of cardiac sympathetic neurotransmitters was associated with increased interstitial norepinephrine (NE) and reactive oxygen species in congestive heart failure (CHF), using a microdialysis technique and salicylate to detect ^.OH generation. Rabbits with and without rapid ventricular pacing (340 beats/min) were randomized to receive desipramine (10 mg/day) or placebo for 8 weeks. Rapid pacing produced left ventricular dilation and systolic dysfunction. The failing myocardium also showed reduced tissue contents of NE and tyrosine hydroxylase protein and activity. In contrast, myocardial interstitial NE was increased in CHF (0.89 ± 0.11 ng/ml) compared to the sham animals (0.26 ± 0.03 ng/ml). In addition, cardiac oxidative stress was increased in CHF as measured by myocardial interstitial ^.OH radical, tissue oxidized glutathione and oxidized mitochondrial DNA. Desipramine treatment produced significant NE uptake inhibition as evidence by an exaggerated pressor response and a greater increase of myocardial interstitial NE in response to intravenous NE infusion, but no significant effects on cardiac function or hemodynamics in sham or CHF animals. However, desipramine treatment attenuated the reductions of tissue NE and tyrosine hydroxylase protein and activity in CHF. Desipramine also prevented the reduction of tyrosine hydroxylase produced by NE in PC12 cells. Thus, the reduction of cardiac sympathetic neurotransmitters is related to the increased interstitial NE and tissue oxidative stress in CHF. Also, normal neuronal uptake of NE is required for NE or its oxidized metabolites to exert their neurotoxic effects.