Myocardial NO signaling appears elevated in heart failure (HF). Whether this results from increased NO production, induction of the high-output NO synthase (NOS)2 isoform, or changes in NOS regulatory pathways (such as caveolae) remains controversial. We tested the hypothesis that increased abundance of caveolin-3 and/or sarcolemmal caveolae contribute to increased NO signaling in pacing-induced HF. Abundance of caveolin-3 (0.59±0.08 versus 0.29±0.08 arbitrary units,P=0.01) but not caveolin-1 was increased in HF compared with control conditions, assessed by Western blot. Additionally, transmission electron microscopy revealed increased caveolae (2.7±0.4 versus 1.3±0.3 per micrometer myocyte membrane,P<0.005). The association between caveolin-3 and NOS3 at the sarcolemma and T tubules was unchanged in HF compared with control myocytes. The impact of NOS inhibition with l-NG-methylarginine hydrochloride (L-NMMA) on &bgr;-adrenergic inotropy was assessed in conscious dogs before and after HF. In control dogs, dobutamine (5 &mgr;g · kg−1· min−1) increased +dP/dt by 36±7%, and this was augmented to 66±24% by 20 mg/kg L-NMMA (P=0.04 versus without L-NMMA, n=8) but not affected by 10 mg/kg L-NMMA (34±10%,P=NS; n=8). In HF, dobutamine +dP/dt response was depressed (P<0.001 versus control), and increased concentrations were required to match control inotropic responses (10 to 15 &mgr;g · kg−1· min−1, 48±7%). L-NMMA enhanced +dP/dt responses similarly at 10 mg/kg (61±17%,P=0.02; n=4) and 20 mg/kg (54±7%,P=0.04; n=7). Caveolin-3 abundance positively correlated with L-NMMA augmentation of dobutamine inotropic responses in HF (r=0.9,P=0.03; n=4). Thus, in canine pacing-induced HF, expression of caveolin-3 and of sarcolemmal caveolae is increased. This increase is associated with augmented agonist-stimulated NO signaling, likely via a compartmentation effect.