Testicular function is highly dependent on temperature control. The aim of this study was designed to investigate the morphological changes and regulation of steroidogenesis by light and electron microscopic level in Leydig cells (LC) after scrotal hyperthermia in rats. The rats were randomly allotted into one of four experimental groups: A (Control), B (1 day after scrotal hyperthermia), C (14 days after scrotal hyperthermia), D (35 days after scrotal hyperthermia); each group contain seven animals. Scrotal hyperthermia was carried out in a thermostatically controlled water bath at 43 degrees C for 30 min once daily for 6 consecutive days. Control rats were treated in the same way, except the testes were immersed in a water bath maintained at 22 degrees C. Hyperthermia applied rats were sacrificed under 50 mg/kg ketamine anaesthesia after 1, 14 and 35 days, and biopsy materials of testis were obtained for light and electron microscopic examinations. To date, no histopathological changes of LC injury after scrotal hyperthermia in rats have been reported. Light microscopic examinations indicated increase degenerative LC, decrease in number of testosterone positive LC in interstitial area after scrotal hyperthermia in short-term. In scrotal hyperthermia, a dilated smooth endoplasmic reticulum, swollen mitochondria, and vanished mitochondrial cristae were observed. The nuclei of some LC displayed deep invaginations and irregular outlines. The number of lipid droplets was very considerably increased in most LC when compared to control group. As a conclusion, we claim that temperatures higher than the body temperature may cause infertility by damaging LC.