Pyrimidine (6-4) pyrimidone photoproduct is the second most abundant UV photoproduct in DNA. Recently, it was reported that Drosophila melanogaster cell-free extracts restored the biological activity of (6-4) photo-product-containing DNA in a light-dependent reaction (Todo, T., Takemori, H., Ryo, H., Ihara, M., Matsunaga, T., Nikaido, O., Sato, K., and Nomura, T. (1993) Nature 361, 372-374) concomitant with the loss of (6-4) photoproduct antigenic sites and (6-4) photoproduct-caused alkali-labile sites. In the present study we show that the (6-4) photoproduct but not its Dewar isomer is the substrate for the enzyme, that the enzyme has an action spectrum peak at 400 nm, and that the efficiency of repair per incident photon is very low compared with cyclobutane pyrimidine dimer photolyases. Furthermore, we provide evidence that the (6-4) photoproduct photolyase converts the photoproduct to unmodified bases probably through an oxetane intermediate.