β-Adrenoceptor/cAMP-dependent Ser¹⁶-phosphoryation as well as Ca²⁺-dependent Thr¹⁷-phosphorylation of phospholamban (PLB) influences SERCA 2a activity and thus myocardial contractility. To determine the cross-signaling between Ca²⁺ and cAMP pathways, the phosphorylation of Ser¹⁶-PLB and Thr¹⁷-PLB was studied at increasing stimulation frequencies as well as in the presence of β-adrenergic stimulation in isolated ventricular trabeculae from failing (dilative cardiomyopathy, DCM, heart transplants, n=9) and non-failing human myocardium (donor hearts, NF, n=9). In addition, we measured the intracellular Ca²⁺-transient (fura-2) at increasing stimulation frequencies (0.5–3.0 Hz). Protein expression of SERCA 2a and phospholamban was similar in DCM and NF. In DCM, diastolic [Ca²⁺]_i was increased and systolic [Ca²⁺]_i as well as Ser¹⁶ PLB-phosphorylation were decreased as compared to NF at 0.5 Hz. The positive force–frequency relationship in human non-failing myocardium was accompanied by a frequency-dependent increase in Ser¹⁶-PLB, but not Thr¹⁷-PLB phosphorylation. In DCM, Ser¹⁶-PLB as well as Thr¹⁷-PLB phosphorylation were not altered at higher stimulation frequencies. After application of isoprenaline (1 µM), a profound increase in Ser¹⁶-PLB phosphorylation was accompanied by a small increase in Thr¹⁷-PLB phosphorylation, only in NF. The frequency-dependent phosphorylation of Ser¹⁶-PLB may favor an increase in Ca²⁺ transient and force generation in humans. Cross talk signaling of Ser¹⁶/Thr¹⁷-PLB phosphorylation after β-adrenergic stimulation exists in non-failing, but not in failing human myocardium. The Ca²⁺-dependent CaM-kinase activity may be altered in human heart failure.