We have previously found that Ca2+ transients decrease at increased stimulation frequency in left atrial (LA) myocytes, but not in right atrial (RA) myocytes. To know underlying mechanism for this difference we investigated SERCA2 and PLB protein expression and their subcellular distributions, and frequency-dependent PLB phosphorylation in rat RA and LA myocytes using Western blot and immunocytochemistry. We found that SERCA2 and PLB monomer expressions were higher in RA than in LA myocytes, and that both of them were more abundant in RA compared to LA myocytes, with peripheral abundance. The expression profile was somewhat consistent with that obtained from total mRNA sequencing in RA and LA tissues from human and rat. Under resting conditions, PLB phosphorylation at both PKA site (Ser16) and CaMKII site (Thr17) were higher in RA than in LA myocytes. In addition, gradual increase in the frequency of electrical stimulations from 0- to 3-Hz in isolated rat RA myocytes increased the level of PLB phosphorylated at Ser16. In contrast, LA did not show any increase in Ser16 phosphorylation. Furthermore, the increased stimulation frequency did not lead to any phosphorylation at Thr17 in either RA or LA myocytes. These results suggest that SR Ca2+ uptake may be more effectively enhanced in RA myocytes in response to increased heart rate, and that PKA may be responsible for the frequency-dependent PLB phosphorylation in RA cells.