A new efficient gene disruption cassette for repeated use in budding yeast has been developed. This cassette, loxP-kanMX-loxP, combines the advantages of the heterologous kan^r marker with those of the Cre-loxP recombination system. The cassette integrates efficiently via homologous recombination at the correct genomic locus (routinely 70%). Upon expression of the Cre recombinase, the kanMX module is excised by recombination between the loxP sites, leaving behind a single loxP site at the chromosomal locus. This system allows repeated use of the kan^r marker gene, which is important for the functional analysis of gene families. The yeast Saccharomyces cerevisiae will be the first eukaryotic organism for which the entire genome sequence will be determined. This will result in the identification of all 6500–7000 genes from this organism. The next challenge is the functional characterization of the unknown gene products. The first step towards a functional analysis of a protein is the complete deletion of the corresponding gene on the chromosome (null mutant) using the one-step gene transplacement method. Classically, DNA fragments flanking the gene of interest are cloned left and right of a yeast marker gene and, after transformation of this construct into yeast, homologous recombination between the flanking regions results in a deletion of the gene and the simultaneous integration of the marker gene. Because yeast has very efficient mechanisms for homologous recombination, it has been possible to reduce the length of the flanking DNA regions to 30–45 bp, allowing the construction of gene disruption cassettes by the polymerase chain reaction (PCR). This system allows construction of gene disruption cassettes without cloning steps and only requires the DNA sequence of the relevant chromosomal locus. The system could be improved significantly by using a heterologous marker gene. This avoids the problem of gene conversion associated with the use of yeast marker genes and recipient yeast strains not completely deleted for the marker gene. The kan^r gene from the Escherichia coli transposon Tn903 when expressed in yeast renders the transformants resistant to the aminoglycoside antibiotic G418. Very recently the kan^r gene was fused to the TEF promoter and terminator sequences from the filamentous fungus Ashbya gossypii yielding the kanMX expression module. PCR-mediated generation of disruption cassettes carrying this completely heterologous expression module allows efficient gene disruptions. An important result from the yeast sequence analysis is the finding that a substantial portion of genes are duplicated in the genome. For example, in silico analysis of the available sequence data revealed the existence of at least 15 proteins belonging to the HXT family of hexose transporters. A second example are the flocculation genes, which constitute a new subtelomeric gene family. ThusA new efficient gene disruption cassette for repeated use in budding yeast has been developed. This cassette, loxP-kanMX-loxP, combines the advantages of the heterologous kan^r marker with those of the Cre-loxP recombination system. The cassette integrates efficiently via homologous recombination at the correct genomic locus (routinely 70%). Upon expression of the Cre recombinase, the kanMX module is excised by recombination between the loxP sites, leaving behind a single loxP site at the chromosomal locus. This system allows repeated use of the kan^r marker gene, which is important for the functional analysis of gene families. The yeast Saccharomyces cerevisiae will be the first eukaryotic organism for which the entire genome sequence will be determined. This will result in the identification of all 6500–7000 genes from this organism. The next challenge is the functional characterization of the unknown gene products. The first step towards a functional analysis of a protein is the complete deletion of the corresponding gene on the chromosome (null mutant) using the one-step gene transplacement method. Classically, DNA fragments flanking the gene of interest are cloned left and right of a yeast marker gene and, after transformation of this construct into yeast, homologous recombination between the flanking regions results in a deletion of the gene and the simultaneous integration of the marker gene. Because yeast has very efficient mechanisms for homologous recombination, it has been possible to reduce the length of the flanking DNA regions to 30–45 bp, allowing the construction of gene disruption cassettes by the polymerase chain reaction (PCR). This system allows construction of gene disruption cassettes without cloning steps and only requires the DNA sequence of the relevant chromosomal locus. The system could be improved significantly by using a heterologous marker gene. This avoids the problem of gene conversion associated with the use of yeast marker genes and recipient yeast strains not completely deleted for the marker gene. The kan^r gene from the Escherichia coli transposon Tn903 when expressed in yeast renders the transformants resistant to the aminoglycoside antibiotic G418. Very recently the kan^r gene was fused to the TEF promoter and terminator sequences from the filamentous fungus Ashbya gossypii yielding the kanMX expression module. PCR-mediated generation of disruption cassettes carrying this completely heterologous expression module allows efficient gene disruptions. An important result from the yeast sequence analysis is the finding that a substantial portion of genes are duplicated in the genome. For example, in silico analysis of the available sequence data revealed the existence of at least 15 proteins belonging to the HXT family of hexose transporters. A second example are the flocculation genes, which constitute a new subtelomeric gene family. Thus