Simian virus 40 minichromosomes were irradiated with ultraviolet light and used to study cell-free DNA repair reactions by soluble extracts of repair-proficient human cells. Damage-specific incorporation of labeled nucleotides was measured after linearization and electrophoresis of the viral DNA. Repair synthesis with irradiated chromosomes was dependent on the dose of ultraviolet light, but naked viral DNA irradiated at the same doses showed significantly higher template activity. The reactions absolutely required the presence of cell extract, but an extract from Chinese hamster cells was as active as those from human cells. The repair activity was apparently stimulated by the addition of unirradiated plasmid DNA, which did not affect the nucleosome density of the viral chromosomes. ATP and its regenerating system were required for the reaction, but total repair synthesis was little affected by exogenous addition of substrates for RNA synthesis and poly(ADP-ribosyl)ation. Most of the repaired viral chromosomal DNA was in the supercoiled, closed circular form, suggesting that excision repair reactions proceeded to the stage of ligation in the present cell-free system.