The cellular mechanism of heat shock-mediated cardioprotection is still under debate. Because heat pretreatment negatively affects the normoxic left ventricular contractile performance in vitro when the extracellular Ca2+ concentration ([Ca2+]o) is relatively low (0.65-1.25 mM), the intracellular Ca2+ homeostasis was studied in more detail in cardiomyocytes isolated from adult rats 24 h after heat stress (42 degrees C for 15 min) or anesthesia (control). Sensitivity to Ca2+ overload was assessed by exposure to veratridine (quiescent cells) or to [Ca2+]o ranging from 0.125 to 20 mM in quiescent and paced cardiomyocytes. The fraction of irreversibly hypercontracted cells was not different between groups. The fura-2 fluorescence ratio (I340/I380), which was used as a measure for cytoplasmic Ca2+ concentration ([Ca2+]i) in quiescent cells after exposure to [Ca2+]o (0.5-10 mM), was also not different between groups. Myofilament Ca2+ sensitivity was assessed in paced (0.5 Hz) cells by simultaneous measurement of [Ca2+]i transients and cell shortening. At stepwise increases of [Ca2+]o from 1 to 10 mM, these parameters were comparable between groups. The diastolic cell length shortened progressively and equally in both groups after increasing [Ca2+]o. However, within 2 min of return from 10 to 1 mM [Ca2+]o, cells from heat-shocked rats retained the same length, whereas cells from control rats contracted further (P = 0.05). These data suggest that heat stress improves relaxation after challenge with high [Ca2+]o.