In a search for a replication complex, the activity that replicates the 2-micrometers yeast DNA plasmid in vitro was isolated in a high molecular weight form (Mr approximately 2 X 10(6) by gel filtration and rate-zonal sedimentation from extracts prepared from cells of the budding yeast Saccharomyces. When obtained from cells in late logarithmic cultures this material or "complex" was labile compared to that from early logarithmic cultures, and it did not survive as a complex after ammonium sulfate precipitation. This suggests that, as cultures approach stationary phase and cells cease growth, the association of its protein constituents may be altered. A chimera of 2-micrometers DNA inserted into the plasmid pBR322 was used to test for binding of components of the complex. After a brief incubation of the chimera in vitro with the high molecular weight material containing replicating activity, a protein "knob" was found associated with the 2-micrometers DNA as shown by electron microscopy. This association was not random but was limited to two positions on the plasmid. In the same series of experiments, the in vitro origins of 2-micrometers plasmid replication were also mapped. Two origins were found, consistent in position with those that have been identified in vivo. Molecules utilizing both origins simultaneously in vitro were not observed, and replication in vitro was bidirectional. The location of the origins corresponded to the positions at which the protein knobs associated with 2-micrometers DNA. This and the fact that no replicative intermediates with associated complexes were detected raises the possibility that a specific protein complex may be involved in initiation of DNA replication.