We have investigated whether mammalian cells can repair pyrimidine dimers in their mitochondrial DNA which have been induced by ultraviolet light. The assay system is based upon the ability of the phage T4 UV endonuclease to nick covalently closed circular mitochondrial DNA that contain pyrimidine dimers. Our results show that dimers are not removed from the mitochondrial DNA of mouse L cells or human KB and HeLa cells. There is also no evidence for photoreactivation of mitochondrial DNA. Analyses of ethidium bromide-cesium chloride equilibrium density gradients of mitochondrial DNA isotopically labeled before and after exposure to ultraviolet light show that the total amount of DNA replication is depressed after exposure. In addition, an increase in the frequency of molecules banding at a position expected for intermediate replicating forms and open circular daughter molecules suggests that the rate of replication is slower (or arrested) in molecules with pyrimidine dimers. The absence of a significant amount of mixing of label incorporated before and after ultraviolet-irradiation is evidence against the occurrence of a large amount of genetic exchange between mitochondrial DNA molecules under these conditions.