The non-transcribed spacer (NTS) regions of the linear extrachromosomal palindromic rDNA from the ciliated protozoan Tetrahymena thermophila contain, in addition to sequences regulating transcription, the origin(s) of bidirectional replication as well as telomere associated sequences. These NTS regions function as autonomous replication sequences (ARS) in the yeast Saccharomyces cerevisiae (Kiss, G.B., Amin, A.A. and Pearlman, R.E., Mol. Cell Biol. 1: 535-543, 1981). We have now identified in the 5' NTS two adjacent but non-overlapping restriction fragments which function as ARS in yeast. These fragments contain 200 bp of duplicated sequence and include the in vivo origin of rDNA replication. The ARS in the 3' NTS has been subdivided into a sequence which allows high frequency transformation of yeast but with transformants extremely unstable when grown either under selective or non-selective conditions, and a sequence which appears to be required for plasmid maintenance yet not to be essential for high frequency transformation. Detailed analysis of the DNA sequences in these regions gives rise to interesting information about structural and functional features of the molecule.