Four sets of plasmid vectors for the budding yeast Kluyveromyces lactis (Kl) have been constructed. All plasmids are pUC19-based shuttle vectors having multiple unique sites in their multiple cloning site (MCS) within the bacterial lacZ gene. The first set of vectors contains Klori, the origin of replication for Kl isolated from Kluyveromyces plasmid pKD1, and one of the selectable nutritional markers, URA3, TRP1 or LEU2. These markers from the yeast, Saccharomyces cerevisiae (Sc), can complement the uraA1, trp1 and leu2 mutations of Kl. The second set of vectors, in addition to Klori, contains the ARS (autonomously replicating sequence) and centromeric sequences of Sc, and are able to replicate in both Sc and Kl. The third group of plasmids is centromeric vectors that are maintained in Kl at low copy number. The last family of vectors was designed for gene overexpression. As they contain the bacterial kanamycin-resistance-encoding gene (kan), plasmid copy number can be amplified to over 100 copies per cell in Kl by growing cells in the presence of the antibiotic G418 (Geneticin). This type of vector has been used to study the high-copy-lethality phenotype of a truncated version of the Kl MGI2 gene encoding the alpha-subunit of the mitochondrial F1F0-ATP synthase.