DNA replication is a highly conserved process essential for the perpetuation of all life forms. Initiation of DNA replication occurs at specific sites called replication origins. The essential DNA elements that constitute these sites are not fully understood in eukaryotes. Studying origins of replication using budding yeasts as a model may provide useful information for deciphering essential components of replication origins in higher eukaryotes, including humans. Origins of replication (also known as autonomously replicating sequences or ARSs) are well characterized in the budding yeast species Saccharomyces cerevisiae. S. cerevisiae ARSs contain an ARS consensus sequence (ACS) that is essential but not sufficient for supporting replication initiation. Little is known about the essential elements of replication origins in other yeast species or in higher eukaryotes. In this study, I have cloned and analyzed the sequence of several ARSs from Saccharomyces kluyveri, and an ARS consensus sequence has been identified, similar to that of S. cerevisiae in that both ACSs are AT-rich 17 bp sequences. Additionally, I have discovered flanking elements that are essential for the function of certain S. kluyveri ARSs. This study provides preliminary data on the essential ACS and a flanking element that constitute functional replication origins in S. kluyveri.