Objective: The protein phosphatase inhibitor-1 (I-1) is a highly specific and potent inhibitor of type 1 phosphatases (PP1) that is active only in its protein kinase A (PKA)-phosphorylated form. I-1 ablation decreases, I-1 overexpression sensitizes β-adrenergic signaling in the heart. It is controversial whether I-1 expression is altered in human heart failure (HF), likely because its detection in heart is difficult due to its low abundance. Methods and results: I-1 was >500-fold enriched from left ventricular myocardium (LVM) from patients with terminal HF (n = 16) and non-failing controls (NF, n = 5) and quantified with an affinity-purified I-1 and a I-1 phosphospecific antiserum. In non-failing I-1 protein levels amounted to 126 fmol/mg protein. In failing hearts, I-1 protein levels were reduced by 58% and I-1 phosphorylation by 77% (P<0.001 vs. NF). I-1 phosphorylation correlated well with serine-16 phosphorylation of phospholamban (PLB) in the same hearts (P<0.001). In contrast, PLB, troponin I (TnI) and PP1 protein and TnI phosphorylation levels did not differ between HF and NF. Conclusions: The results suggest that the reduction in I-1 protein and phosphorylation in failing human hearts leads to increased phosphatase activity which in turn may result in reduced phosphorylation of cardiac proteins such as PLB.