Studies of UV-induced skin cancers show that malignisation of skin cells, as well as alterations in anti-tumor immune control, are triggered by UV-induced lesions in cellular DNA. Such lesions can probably appear in the human mononuclear leukocytes (lymphocytes) during exposure of skin to sunlight. With the aim of studying the processing of UV-induced DNA lesions in these cells, we used flow cytometry and labelling of their partially denatured nuclei with the monoclonal antibody (H3) that binds cyclobutane pyrimidine dimers in single-stranded DNA. After the first few hours of cultivation of the irradiated cells, we found an increase in H3-specific fluorescence from cellular nuclei, while there was a decrease in the number of H3-positive sites in isolated DNA from these cells. We examined cells cultured under different conditions and concluded that the effect of enhancement of H3 labelling of nuclei did not result from changes in temperature and culture medium. Furthermore, we have found that this effect, as well as the decrease in H3 labelling in isolated DNA, are both prevented by pretreatment of the cells with Novobiocin, which we used as an inhibitor for the topoisomerase II-induced relaxation of supercoiled DNA prior to repair-specific incision. The inhibition by Novobiocin of the above-mentioned changes in H3 labelling in cellular nuclei and isolated DNA of the irradiated cells clearly indicate the association of both effects with an excision repair-related DNA modification. While the partial loss of H3-binding sites from isolated DNA is obviously a result of excision of some fraction of pyrimidine dimers, the enhancement of the H3 labelling of nuclei might be due to the formation of open structures at dipyrimidine-containing DNA fragments in preparation for incision. We suggest that formation of open structures predominates quantitatively over dual incision and excision of these fragments, and leads to enhanced exposure of the pyrimidine dimers in nuclei to H3 binding. Thus, unstimulated human lymphocytes appear to be capable of performing pre-incision steps for removal of these DNA lesions. © The Royal Society of Chemistry and Owner Societies 2004.