The cardiac sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA2) participates in termination of myocyte contraction by transferring cytoplasmic Ca²⁺ ions into the lumen of sarcoplasmic reticulum. We used immunoblotting with a polyclonal anti-SERCA antibody to quantify SERCA2 expression in atrium and ventricle of 3 teleost fish species, burbot (Lota lota), rainbow trout (Oncorhynchus mykiss) and crucian carp (Carassius carassius) acclimated to low (4°C, cold-acclimated, CA) or high (18°C, warm-acclimated, WA) temperature. SERCA2 expression was over 4 times higher (P <0.05) in the heart of CA than WA burbot (in both cardiac chambers), while no differences were observed in SERCA2 expression between atrium and ventricle (P >0.05). In the rainbow trout heart, SERCA2 expression was 6- (P <0.05) and 23-fold (P <0.001) higher in atrial than ventricular muscle for WA and CA fish, respectively. Acclimation to cold increased SERCA2 in atrium (P <0.05), but not in ventricle (P >0.05). In the heart of crucian carp, no thermal responses were observed in the SERCA2 expression. However, atrio-ventricular differences existed in the isoform composition of SERCA2. In both cardiac chambers two SERCA2 isoforms were present. However, in the ventricle, the smaller (93 kDa) isoform was dominating (82–88%), while the larger (110 kDa) isoform represented only 12–18% (P <0.05) of the total SERCA2 protein. In the atrial tissue, both isoforms were almost equally expressed. These results show that in the cold, SERCA2 is up-regulated in the hearts of cold-active teleosts, burbot and rainbow trout, while it remains unaltered in the heart of cold-dormant crucian carp. Chamber-specific differences in the SERCA2 isoform composition exists in the heart of rainbow trout and crucian carp, but not in burbot.