Studies on the status of multifunctional Ca²⁺-calmodulin (CaM)-dependent protein kinase-II (CaMKII) in failing hearts are limited and controversial. The study was performed in the left ventricular (LV) myocardium of six dogs with heart failure (HF) (LV ejection fraction, 23 ± 2%) and six normal (NL) dogs. In the LV homogenate, CaMKII activity and its protein level were determined by using the CaMKII peptide and antibody, respectively. Furthermore, the protein level of CaM and phosphorylated phospholamban (PLB) at threonine-17 (PLB-Thr¹⁷) and serine-16 (PLB-Ser¹⁶) were also determined in the LV homogenate using a specific antibody. In addition, the level of zinc, which inhibits protein kinase A activity, was determined in the LV tissue by inductively coupled plasma mass spectrometry. CaMKII activity and phosphorylated PLB-Thr¹⁷ and PLB-Ser¹⁶ levels, but not CaM and Zn levels, were significantly reduced in the LV homogenate of dogs with HF compared with NL dogs. These results suggest that CaMKII activity is reduced in the failing LV myocardium, and this abnormality is associated with reduced protein expression level of the enzyme but not due to changes in CaM and zinc levels. In conclusion, reduced CaMKII activity and phosphorylated PLB level may be partly responsible for impaired sarcoplasmic reticulum function in HF.