Photolyases are DNA repair enzymes that use energy from blue light to repair pyrimidine dimers. We report the isolation of an Arabidopsis thaliana mutant (uvr2-1) that is defective in photorepair of cyclobutylpyrimidine dimers (CPDs). Whereas uvr2-1 is indistinguishable from wild type in the absence of UV light, low UV-B levels inhibit growth and cause leaf necrosis. uvr2-1 is more sensitive to UV-B than wild type when placed under white light after UV-B treatment. In contrast, recovery in darkness or in light lacking photoreactivating blue light results in equal injury in uvr2-1 and wild type. The uvr2-1 mutant is unable to remove CPDs in vivo, and plant extracts lack detectable photolyase activity. This recessive mutation segregates as a single gene located near the top of chromosome 1, and is a structural gene mutation in the type II CPD photolyase PHR1. This mutant provides evidence that CPD photolyase is required for plant survival in the presence of UV-B light.