Abstract—The effects of singlet oxygen (1O2), generated by the thermal decomposition of water soluble NDPO2(endoperoxide of the disodium 3,3'‐(1,4‐naphthylidene) dipropionate), on a single‐stranded shuttle vector were analysed.1O2induces a much higher level of breaks in the phosphodiester backbone of single‐stranded than double‐stranded DNA. This may be due to a higher accessibility of guanine residue, primarily damaged by1O2. The damaged vector was transfected into monkey COS7 cells where single‐stranded DNA was converted to the double‐stranded replicative form DNA. After 3 days, extrachromosomal DNA was extracted and the plasmids rescued inE. colito study mutagenesis. There is a significant increase in mutation frequency of damaged single‐stranded DNA in comparison to untreated DNA. It is concluded that1O2induces breaks in the backbone of single‐stranded DNA and that the1O2‐damaged molecules are mutated after passage th