Hybrid plasmids containint the bacterial resistance-transfer factor pBR322 and the yeast leu2+ gene have been used to isolate DNA fragments of Physarum that are capable of initiating DNA replication in a yeast host. Five of forty hybrid plasmids containint Physarum sequences transform leu2- yeast to Leu+ at high frequency. The resulting Leu+ transformants are characterized by phenotypic instability. Supercoiled plasmid molecules containing pBR322 sequences can be detected in the transformed yeast, indicating that the transforming DNA replicates autonomously. Plasmid DNA isolated from Leu+ yeast can transform leuB bacteria. The hybrid plasmid recovered from the Leu+ bacterial transformants is identical to the original plasmid, indicating structural integrity is maintained during passage through the yeast host. These hybrid plasmids containing Physarum sequences have the same characteristics as those containing autonomously replicating yeast chromosomal sequences. As the temporal sequence of DNA replication is particularly accessible to study in Physarum plasmodia, the functional significance of these segments should be amenable to study.