Heat acclimation (AC) improves cardiac mechanical and metabolic performance. Using cardiomyocytes and isolated hearts from 30d and 2d acclimated rats (AC, AC-2d, 34°C), we characterized cellular contractile mechanisms under normothermic (37°C) and hyperthermic (39°C-42°C) conditions. To determine contractile responses, Ca²⁺ transients (Ca²⁺T), sarcoplasmic reticulum (SR) Ca²⁺ pool size (Fura-2/Indo-1 fluorescence), force generation (amplitude systolic motion-ASM), L-type Ca²⁺ channels (dihydropyridine receptor-DHPR), Ryanodine receptors (RyRs) and total and phosphorylated phospholamban (PLBt,s,tr), proteins and transcripts, were measured (Western blot, RT-PCR). Cardiac mechanical performance was measured using Langendorff system. We demonstrated that AC/AC-2d increased Ca²⁺T amplitude (148%/147%) and twitch force (180%/130%) and desensitized myofilaments, as indicated by a rightward shift in the ASM-calcium relationships, despite no change in SR Ca²⁺ pool size. Hence, generation of higher Ca²⁺T underlies greater force development in AC/AC-2d myocytes. In isolated hearts, Ryanodine administration eliminated differences between AC and C hearts, implying an important role for RyRs in that acclimation phase. Increased expression of DHPR, RyRs and decreased PLBs/PLBt in AC hearts only, suggest that different pathways increase force generation in the AC-2d vs AC myocytes. At basal beating rates, hyperthermia (39-41°C) enhanced pressure generation in AC hearts. C hearts failed to restitute pressure beyond 39°C. Increased beating frequency produced negative inotropic response. In C cardiomyocytes, hyperthermia elevated basal cytosolic Ca²⁺ and tension, Ca²⁺T and ASM. AC myocytes enhanced Ca²⁺T but showed myofilament desensitization, suggesting its involvement in cardiac protection against hyperthermia. Collectively, both Ca²⁺ turnover and myofilaments responsiveness are important adaptive acclimatory targets during normothermic and hyperthermic conditions.