We investigated whether biventricular (BiV) pacing favourably affects cardiac sympathetic activity in heart failure (HF). In 10 HF patients treated with BiV pacing, we assessed cardiac sympathetic activity by metaiodobenzylguanidine ((123)I-MIBG) imaging. Patients were randomized in a double-blinded crossover fashion, for two weeks of either inactivation of BiV pacing or BiV pacing, with crossover to the alternate group for a further two weeks. After randomization blocks, cardiac (123)I-MIBG imaging and a 6 min walk test were performed. BiV pacing was associated with significant improvements in cardiac (123)I-MIBG uptake reflected by increases in early (BiV 1.71 +/- 0.09 vs. non-BiV 1.63 +/- 0.06, P = 0.03) and late (at 4 h) heart to mediastinal ratio of uptake (BiV 1.54 +/- 0.08 vs. non-BiV 1.45 +/- 0.06, P = 0.03). Additionally, pulmonary (123)I-MIBG uptake, measured as lung to mediastinal ratio, significantly improved (P = 0.009). Six-minute walk and systolic blood pressure tended to improve with BiV vs. non-BiV pacing (P = 0.09). In patients with stable HF, BiV pacing is associated with long-term improvements in cardiac sympathetic nerve activity, as reflected by improvements in cardiac (123)I-MIBG uptake. This is a potential mechanism for morbidity and mortality benefits observed in larger studies.