A hyperimmune rabbit antiserum to ultraviolet-irradiated DNA was observed to contain two activities directed against irradiated DNA. Radioimmunoassay studies demonstrated that both antigenic determinants were pyrimidine dimers as evidenced by the reduction in antibody binding to enzymatically photoreactivated irradiated DNA. Enzymatic photoreactivation specifically monomerizes pyrimidine dimers in DNA. Under conditions in which antiserum binding was measured with native DNA, binding was observed with DNA that contained both thymine-thymine and cytosine-containing dimers but not against native DNA that contained only thymine-thymine dimers. Under these assay conditions, competitive binding to irradiated DNA increased as a linear function of fluence of ultraviolet radiation. Under a second set of conditions in which binding was measured with single-stranded DNA, antibodies were bound to DNA which contained only thymine-thymine dimers. The fluence response for the increase in binding to thymine-thymine dimers was nonlinear and increased as a function of fluence squared. Binding to thymine dimers appeared to require bivalent attachment to two dimers that occurred in close proximity.