ABSTRACr DNA fragments of Xenopus laevis, the African frog, were cloned in the EcoRI site of the Escherichia coli Rlasmid pACYC189 and tested for ability to initiate and complete replication of the recombinant plasmid when injected into unfertilized eggs of X. laevis. After measurement of the [3HJ thymidine incorporation per egg for a number of recombinant plasmids, pSW14 and pSW9, which respectively contain a small segment (5SO base pairs) and several kilobases of frog DNA, were selected for more extensive analysis. In spite of the small size of the segment in pSW14, it incorporates in 2 hr at least 3 times as much labeled thymidine as either pSW9 or the vector alone. The pNA synthesis in pSW14 was shown to be replication rather than repair synthesis, based on a buoyant density shift of the product when iododeoxyuridine was used for labeling. To determine the number of replications of pSW14, a novel mpethod was employed. Because pSW14 is a head-to-head dimer of the vector with the Xenopus fragment inserted at an EcoRI site, the plasmid has three methylatable sites-two bracketing (4) in which the S phase is 6-7 hr. In some organisms, cells of the early embryo complete the S phase in a few minutes, and many more functional origins have been detected in Drosophila, for example (5). The time interval between cleavages in sea urchin embryos and some amphibian embryos is so short that origins would have to be close together and operating simultaneously to allow completion of replication in the time observed. Callan (6), by the use of DNA fiber autoradiography, has estimated that the interval in Triturus vulgaris (a salamander) might be as small as 10Mim (30,000 bp). However, a study by DNA fiber autoradiography of Chinese hamster (CHO) cells in culture indicated that potentially functional origins might be as close as 4 jm (12,000 bp) in the DNA that replicates early in S phase (7).With these studies in mind we initiated studies on the cloning and characterization of the origins in Xenopus laevts (the African clawed frog), which has been widely used to study the expression (transcription and translation) of foreign genes in oocytes (8-12). It also has been shown that unfertilized X. laevt eggs have the ability to support DNA replication from injected exogenous DNA (13)(14)(15).A number of derivatives of pACYC189 containing segments Escherichia coil SF8 (rjm-lop-1l, rec B-, rec C-) and plasmid pACYC189 were provided by S. N. Cohen through the Plasmid Reference Center (Stanford University). Plasmid pBR322 and E. colh RY13 were obtained from H.