Pyrimidine dimer production and excision were studied in ultraviolet light (UV)-irradiated primary cultures of epidermal cells derived from perinatal mouse skin and in an in vitro malignantly transformed epidermal cell line. Dimer production increased linearly with UV dose level for both cell types. However, at any given UV dose level, there were 20% fewer thymine-containing dimers induced in the primary cultures compared to the transformed cell line. The reduced dimer yield in the primary cultures was attributed to the multilayer structure (three cell layers) of these cultures. The primary cultures were found to excise no more than 10% of the original dimers in a 24-hr period, while the malignantly transformed cells excised 34%. Nonsemiconservative DNA repair synthesis was also studied as a function of dimer yields in the first 3 hr after irradiation. When the levels of repair replication in both cell types were compared at equal yields of UV-induced dimers, the malignantly transformed cells exhibited a higher level of repair than did the primary epidermal cells. There was no difference in the kinetics of repair replication between the two cell types at a UV dose level of 10 J/sq m over the first 6 hr after irradiation.