We characterized [methyl-³H]thymidine ([³H]thymidine) and [5-³H]uridine ([³H]uridine) incorporation into cultured astrocytes and neurons in the presence and absence of hydrogen peroxide (H₂O₂) in order to define the response to oxidative stress in the central nervous system. [³H]Thymidine incorporation into cultured astrocytes was remarkably decreased by N⁶,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP), a permeable analogue of cAMP, which induced a morphological change from the polygonal form (undifferentiated astrocytes) to the process-bearing one (differentiated astrocytes). H₂O₂ induced [³H]thymidine, but not [³H]uridine, incorporation into cultured astrocytes at only an early time from 24 h after DBcAMP treatment, although the absolute quantities of [³H]thymidine incorporation into astrocytes pretreated with DBcAMP were less than those into astrocytes pretreated without DBcAMP. Hydroxyurea, a replicative DNA synthesis inhibitor, suppressed dose-dependently and completely [³H]thymidine incorporation into astrocytes pretreated without DBcAMP, but not astrocytes pretreated with DBcAMP. H₂O₂ did not stimulate [³H]thymidine or [³H]uridine incorporation into astrocytes pretreated without DBcAMP and neurons. These findings indicate that only astrocytes pretreated with DBcAMP are able to increase thymidine incorporation specifically in the presence of H₂O₂ for a purpose other than proliferation, including the repair of H₂O₂-induced DNA injury, for example.