In the present investigation a comparison was made between conventional histology, histochemistry and vital microscopy for assessment of heat-induced bone tissue injury. The extent of the bone damage around a burr hole or after heating fibular bone samples in saline solutions of various temperatures was evaluated by means of histology and histochemistry, using the absence of filled osteocyte lacunae or lack of oxidative enzyme activities as indication of bone death, respectively. In both cases a wider necrotic border zone was detected with histochemistry than with histology. The vital microscopic method is based on a titanium chamber which, after insertion in the rabbit tibia, allows observation and registration of the same bone compartment for a follow-up period of more than one year. The dynamic tissue events taking place after heating to 50 degrees C for one minute were investigated and compared with histological and histochemical data of the bone. The vital microscopic method showed a consistent and widespread bone tissue injury after heating to 50 degrees C for one minute while, on the other hand, the indirect methods exhibited only inconsistent signs of tissue injury. It is concluded that histochemistry using the presence or otherwise of diaphorase enzyme activities is a more reliable method than is histology for the estimation of bone viability after heat trauma. Vital microscopy is more sensitive than indirect, morphologic and metabolic techniques for detection of heat-induced bone tissue injury.