Endosymbiotic transfer of DNA and functional genes from the cytoplasmic organelles (mitochondria and chloroplasts) to the nucleus has been a major factor driving the origin of new nuclear genes, a process central to eukaryote evolution. Although organelle DNA transfers very frequently to the nucleus, most is quickly deleted, decays, or is alternatively scrapped. However, a very small proportion of it gives rise, immediately or eventually, to functional genes. To simulate the process of functional transfer, we screened for nuclear activation of a chloroplast reporter gene aadA, which had been transferred from the chloroplast to independent nuclear loci in 16 different plant lines. Cryptic nuclear activity of the chloroplast promoter was revealed, which became conspicuous when present in multiple nuclear copies. We screened ∼50 million cells of each line and retrieved three plants in which aadA showed strong nuclear activation. Activation occurred by acquisition of the CaMV 35S nuclear promoter or by nuclear activation of the native chloroplast promoter. Two fortuitous sites within the 3′ UTR of aadA mRNA both promoted polyadenylation without any sequence change. Complete characterization of one nuclear sequence before and after gene transfer demonstrated integration by nonhomologous end joining involving simultaneous insertion of multiple chloroplast DNA fragments. The real-time observation of three different means by which a chloroplast gene can become expressed in the nucleus suggests that the process, though rare, may be more readily achieved than previously envisaged. ; Andrew H. Lloyd and Jeremy N. Timmis