A method for the simultaneous integration of two or more copies of a polynucleotide of interest into the chromosome of a fungal host cell, said method comprising the steps of: (a) providing a fungal host cell comprising in its chromosome at least two integration sites, each integration site comprising a pair of recognition sequences of a site-specific recombinase, each pair flanking a resident negative selection marker, wherein the site-specific recombinase and its pair of recognition sequences are from the FLP-FRT system of Saccharomyces cerevisiae and wherein the negative selection marker encodes a polypeptide having cytosine deaminase activity; (b) introducing into said cell a nucleic acid construct comprising a pair of recognition sequences of the site-specific recombinase, said pair flanking the polynucleotide of interest; (c) transiently expressing the site-specific recombinase in the cell, whereby the chromosomal recognition sequence pairs are recombined with the corresponding recognition sequence pair of the nucleic acid construct by the recombinase, so that at the least two integration sites, the resident negative selection marker in the chromosome is excised while a copy of the polynucleotide of interest is integrated in its place to produce a fungal host cell comprising two or more copies of the polynucleotide of interest integrated into the chromosome of the fungal host cell; and (d) cultivating the fungal host cell of step (c) in a selective medium comprising sufficient amounts of 5-fluorocytosin to be converted to an inhibitory concentration of toxic 5-fluorouracil by said cytosine deaminase, and selecting a cell in which the fungal host cell comprises at least two polynucleotides of interest integrated into the chromosome of the fungal host cell. (a) providing a fungal host cell comprising in its chromosome at least two integration sites, each integration site comprising a pair of recognition sequences of a site-specific recombinase, each pair flanking a resident negative selection marker, wherein the site-specific recombinase and its pair of recognition sequences are from the FLP-FRT system of Saccharomyces cerevisiae and wherein the negative selection marker encodes a polypeptide having cytosine deaminase activity; (b) introducing into said cell a nucleic acid construct comprising a pair of recognition sequences of the site-specific recombinase, said pair flanking the polynucleotide of interest; (c) transiently expressing the site-specific recombinase in the cell, whereby the chromosomal recognition sequence pairs are recombined with the corresponding recognition sequence pair of the nucleic acid construct by the recombinase, so that at the least two integration sites, the resident negative selection marker in the chromosome is excised while a copy of the polynucleotide of interest is integrated in its place to produce a fungal host cell comprising two or more copies of the polynucleotide of interest integrated into the chromosome of the fungal host cell; and (d) cultivating the fungal host cell of step (c) in a selective medium comprising sufficient amounts of 5-fluorocytosin to be converted to an inhibitory concentration of toxic 5-fluorouracil by said cytosine deaminase, and selecting a cell in which the fungal host cell comprises at least two polynucleotides of interest integrated into the chromosome of the fungal host cell.