Studies were initiated to investigate whether mechanisms exist within mitochondria to repair damage incurred by mitochondrial DNA after exposure to alkylating toxins. A clonal isolate from a rat insulinoma cell line was utilized to measure the formation and repair of alkali-labile sites within the mitochondrial genome after exposure to the alkylating antibiotic streptozotocin. Alkali-labile sites were formed in mitochondrial DNA in a dose-dependent fashion. Eight hours after exposure to the toxin, 55% of the lesions were removed. The level of repair increased to 70% after 24 h. In comparison, only 46% of N7-methylguanines were removed across the entire cellular genome. These studies demonstrate that streptozotocin causes appreciable mitochondrial DNA damage in a dose-dependent manner and provide the first evidence that a repair mechanism for alkali-labile sites is present within the mitochondrion.