Cytogenetic and molecular analyses were performed in G1 Chinese hamster ovary cells treated with 254 nm UV light, to test the hypothesis that UV may affect the recognition site of specific restriction endonucleases (RE). Since short-wavelength UV light induces mainly cyclobutane dimers (CPD) at TT and CT sequences, RE were selected according to the presence or absence of thymine at the recognition site (DraI TTT/AAA, AluI AG/CT and HaeIII GG/CC). A drastic reduction of DraI-and AluI-induced chromosome-type aberrations was found in cells pretreated with UV. Conversely, such a reduction was not observed with HaeIII. To better understand this phenomenon, a molecular analysis was carried out at both the genome level and at the hypoxanthine phosphoribosyl transferase gene level, showing that the cutting pattern of DraI on isolated DNA from UV-irradiated cells was strongly reduced compared with an untreated sample, whereas HaeIII was not able to modify the cutting pattern of irradiated cells. Our data demonstrate a good correlation between the results obtained with cytogenetic and molecular approaches, suggesting that cyclobutane dimers are the main lesions responsible for the observed reduction of the cleaving activities of RE, at both the chromatin and naked DNA levels.