Heat shock protein 70 (Hsp70) can physically interact with and prevent thermal inactivation of SERCA1a, the SERCA isoform expressed in adult fast-twitch skeletal muscle. This study examined whether Hsp70 could physically interact with and prevent thermal inactivation of SERCA2a, the SERCA isoform expressed in heart. HEK-293 cells were co-transfected with cDNAs encoding human Hsp70 and rabbit SERCA2a (S2a/Hsp70). Cells co-transfected with SERCA2a cDNA and pMT2 (S2a/pMT2) were used as control. Half of the cells were heat shocked at 40°C for 1 hr (HS) and half were maintained at 37°C prior to harvesting the cells and isolating microsomes. Western blot analysis showed that Hsp70 and SERCA2a were co-localized in the microsomal fraction. The levels of Hsp70 were ~5-fold higher (p<0.05) in S2a/Hsp70 compared with S2a/pMT2 and ~2-fold higher (p<0.05) following HS in all cells. Co-immunoprecipitation demonstrated that Hsp70 directly binds to SERCA2a. Following HS, maximal SERCA2a activity was reduced (~52%, p<0.05) in S2a/pMT2 but was increased (~33%, p<0.05) in S2a/Hsp70. Thermal inactivation of SERCA2a in S2a/pMT2 was associated with decreased (~49%, p<0.05) binding capacity for FITC and increased carbonyl (~42%, p<0.05) and nitrotyrosine (~40%, p<0.05) levels in SERCA2a. By contrast, the HS-induced increase in maximal SERCA2a activity observed in S2a/Hsp70 corresponded with no change (p>0.05) in FITC binding capacity and reductions in carbonyl (~40%, p<0.05) and nitrotyrosine (~23%, p<0.05) levels in SERCA2a compared with S2a/pMT2. These results show that Hsp70 forms a protective interaction with SERCA2a during heat stress actually reducing oxidation and nitrosylation of SERCA2a thus increasing its maximal activity.