—The β₂-adrenergic receptor (β₂AR) exists in multiple polymorphic forms with different characteristics. Their relevance to heart failure (HF) physiology is unknown. Cardiopulmonary exercise testing was performed on 232 compensated HF patients with a defined β₂AR genotype. Patients with the uncommon Ile164 polymorphism had a lower peak V̇o₂ (15.0±0.9 mL · kg⁻¹ · min⁻¹) than did patients with Thr164 (17.9±0.9 mL · kg⁻¹ · min⁻¹, P<0.0001). The percentage achieved of predicted peak V̇o₂ was also lower in patients with Ile164 (62.3±4.5% versus 71.5±5.1%, P=0.045). The relative risk of a patient having a V̇o₂ ≤14 mL · kg⁻¹ · min⁻¹ who had Ile164 was 8.0 (P=0.009). Catheterization-based invasive exercise testing revealed depressed changes in the exercise-induced cardiac index, systemic vascular resistance, stroke volume, and V̇o₂ in patients with Ile164. The polymorphisms at position 16 also impacted exercise capacity: peak V̇o₂ for Arg16 versus Gly16 was 17.0±0.8 versus 15.6±0.5 mL · kg⁻¹ · min⁻¹, respectively (P=0.03). Because the polymorphisms at loci 16 and 27 can occur together, 4 homozygous combinations exist. Patients with Arg16/Glu27 had the highest percentage achieved of predicted peak V̇o₂ (75.7±6.4%), whereas those with Gly16/Gln27 had the lowest (55.3±2.8%, P=0.0032). The above findings were not confounded by baseline clinical characteristics, including β-blocker usage. We conclude that the β₂AR polymorphisms Ile164, Gly16, and the combination of Gly16 and Gln27 are associated with depressed exercise performance in HF and represent a genetically determined factor in the pathophysiology of HF.