The survival of M13 DNA was studied after partial replacement of thymine by uracil in the bacteriophage. Uracils carry the same genetic information as the thymines. Nevertheless in Escherichia coli wild-type cells, uracils in DNA are replaced by thymines by excision repair initiated by uracil-DNA glycosylase (UDG). Thus inactivation of uracil-containing phage DNA is solely due to repair initiated by UDG. Incorporation of uracils was achieved in one or in both strands, either randomly or site-specifically using differently uracylated oligonucleotides. The results show that up to 580 uracils can be repaired without a significant decrease in survival if the uracils are localized in the (-) strand only. Incorporation of 246 uracils into the (+) strand leads to approximately 30% or approximately 10% survival when expressed in Escherichia coli strains CMK and JM103, respectively. However, when uracils are distributed over both strands a sharp decrease in survival occurs. This shows that the repair of two uracils localized in close proximity on opposite strands of the DNA by the excision repair mechanism is difficult, whereas uracils occurring in one strand are repaired more efficiently, irrespective of their number.