AbstractWe have undertaken a search for autonomously replicating (ARSs) fromKluvermyces lactischromosomal DNA able to sustain plasmid replication inK. lactisand inSaccharomyces cerevisiae. The discovery of such sequences might be interesting for the comparison of ARSs from different sources and possibly useful for the construction of multivalent vectors.HindIII fragments fromK. lactischromosomal DNA were inserted in the YIp5 plasmid (lacking an origin of replication) and the resulting chimaeric plasmids were selected for the ability to transformS. cerevisiae. Four plasmids were identified and further analysed. Two contained the same 1.8 kbK. lactisfragment and transformed bothK. lactisandS. cerevisiaewith the same efficiency and stability, whereas the third transformed onlyS. cerevisiaeand the fourth transformedK. lactiswith a higher efficiency thanS. cerevisiae.A detailed study was performed on the 1.8kb fragment which exhibited ARS function in both yeasts. The fragment was subcloned using different restriction enzymes andBal31 exonuclease. Subclones were tested for ARS function. ARS activities in the two yeasts were localized in the same 100 bp region. Sequencing demonstrated the presence in this region of the dodecanucleotide 5′ATTTATTGTTTT 3′ differing from the ARS core consensus ofS. cerevisiaeonly by a T insertion.A similar nucleotide sequence ofS. cerevisiaeonly in the putative replication origin of the 2 μ‐like plasmid pkD1 which stably replicates inK. lactis. Homologies with ARSs fromS. cerevisiaewere also found in the regions flanking the above‐mentioned dodecanu