Streptococcus species release cholesterol-dependent cytolysins (CDCs), which are a main toxin, and their heat susceptibility is poorly understood. The aim of this study was to clarify the heat susceptibility of streptococcal exotoxins and CDCs. Streptococcal exotoxins were treated with heat incubation at 60 degrees C for 10 or 30 min. The Streptococcus suis exotoxin of serotypes 1 and 2 exhibited more than 50 % haemolytic activity, and all Streptococcus pneumoniae exotoxins exhibited more than 60 % haemolytic activity. During the thermolabile assay, the virulent streptococcal haemolytic activity remarkably decreased after being heated at 60 degrees C for 10 m. Then, streptococcal recombinant CDCs were produced and put through a thermolabile assay. The haemolytic activity of suilysin (SLY), pneumolysin (PLY) and streptolysin O (SLO) decreased more than 80 % after heat incubation. We also conducted a TER assay to evaluate the cell monolayer. The cell monolayer of all CDCs broke down, and the FITC-dextran translocated at 1 h post addition, while the CDCs treated with heating did not induce cell disruption. Moreover, the microscopy analysis demonstrated that CDCs treated with heating lost their activity. In conclusion, heat incubation induced the inactivation of streptococcal exotoxins and CDCs. Heat incubation plays a role in the degradation of the streptococcal exotoxin, and this result applies to the inhibition of streptococcal infection.