A hybrid cell line (hybridoma) has been isolated after fusion between mouse‐plasmacytoma cells and spleen cells from mice immunized with a thymine dimer‐containing tetranucleotide coupled to a carrier protein. Monoclonal antibodies produced by this hybridoma were characterized by testing the effect of various inhibitors in a competitive enzyme‐linked immunosorbent assay (ELISA). The antibodies have a high specificity for thymine dimers in single‐stranded DNA or poly(dT), but do not bind UV‐irradiated d(TpC)₅. Less binding is observed with short thymine dimer‐containing sequences. In vitro treatment of UV‐irradiated DNA with photoreactivating enzyme in the presence of light, or with Micrococcus luteus UV‐endonuclease results in disappearance of antigenicity. Antibody‐binding to DNA isolated from UV‐irradiated human fibroblasts (at 254 nm) is linear with dose. Removal of thymine dimers in these cells during a post‐irradiation incubation, as detected with the antibodies, is fast initially but the rate rapidly decreases (about 50% residual dimers at 20 h after 10 J/m²). The induction of thymine dimers in human skin irradiated with low doses of UV‐B, too, was demonstrated immunochemically, by ELISA as well as by quantitative immunofluorescence microscopy.