A strain of Saccharomyces cerivisiae carrying a mutation in the URA1 gene was transformed with fragments of Dictyostelium DNA inserted into a plasmid capable of replication in E. coli or yeast. Rare prototrophic colonies were recovered that all contained the parent plasmid and an insert of Dictyostelium DNA. Resistance to the antibiotic G418, a function coded by the same plasmid, was also expressed in the prototrophs. Plasmids recovered from the prototrophic yeast could be used to transform E. coli. The E. coli transformants harbored plasmids capable of transforming yeast URA1 mutants to prototrophy. No complementation of the E. coli pyrD mutation, which corresponds to URA1, occurred. Southern blot analysis revealed that the insert of Dictyostelium DNA contained a unique sequence of 1700 base pairs and a repetitive one of 1000 base pairs. Subcloning experiments showed that only the unique sequence was required for complementation, which is independent of the orientation of the Dictyostelium sequence in the plasmid. The repetitive fragment was not linked to the unique sequence in the genome and was probably an artifact of the ligation procedure. The unique sequence hybridized to a Dictyostelium polyA+ RNA species of 1200-1300 bases.