Damaged DNA-binding protein (DDB) is a heterodimer (DDB1 and DDB2), which is implicated in the repair of UV-irradiated DNA damage. Here we have identified four DDB2 variants from HeLa cells (D1-D4) that are generated by alternative splicing. Analysis of tissue distribution by RT-PCR indicates that D1 is the most highly expressed in human brain and heart. A DNA repair assay revealed that both D1 and D2 are dominant negative inhibitors. Electrophoresis mobility shift assays indicated that D1 and D2 are not part of the damaged DNA-protein complex. Co-immunoprecipitation studies show that DDB2-WT interacts with D1 and itself. Nuclear import of DDB1 was less induced by transfection with D1 than WT. Based on these results, D1 and D2 are dominant negative inhibitors of DNA repair, which is probably due to disruption of complex formation between DDB1 and DDB2-WT and of DDB1 nuclear import.