Enhanced DNA repair is an important factor in drug resistance in cancer. Using cell-free extracts derived from the fission yeast, Schizosaccharomyces pombe, we demonstrate in an in vitro system DNA repair system that increased cAMP levels, which activates cAMP-dependent protein kinase (PKA), inhibits repair of ultraviolet (UV)-damaged DNA. Supplementing the cell-free system with the catalytic kinase subunit of PKA also inhibits DNA repair. In contrast, addition of the PKA inhibitor H-89 enhances repair activity. These results show that PKA regulates DNA repair synthesis, thus implicating the cAMP signaling pathway in DNA damage response and repair of UV-damaged DNA lesions.