Perturbations of Simian Virus 40 (SV40) DNA replication by ultraviolet (UV) light during the lytic cycle in permissive monkey CV-1 cells resemble those seen in host cell DNA replication. Formation of Form I DNA molecules (i.e. completion of SV40 DNA synthesis) was more sensitive to UV irradiation than synthesis of replicative intermediates or Form II molecules, consistent with inhibition of DNA chain elongation. The observed amounts of [3H]thymidine incorporated in UV-irradiated molecules could be predicted on the assumption that pyrimidine dimers are responsible for blocking nascent DNA strand growth. The relative proportion of labeled Form I molecules in UV-irradiated cultures rapidly increased to near-control values with incubation after 20 or 40 J/m2 of light (0.9--1.0 or 1.8--2.0 dimers per SV40 genome, respectively). This rapid increase and the failure of Form II molecules to accumulate suggest that SV40 growing forks can rapidly bypass many dimers. Form II molecules formed after UV irradiation were not converted to linear (Form III) molecules by the dimer-specific T4 endonuclease V, suggesting either that there are no gaps opposite dimers in these molecules or that T4 endonuclease V cannot use Form II molecules as substrates.