We report on the use of a new homologous marker for use in multiple gene deletions in S. cerevisiae, the general amino acid permease gene (GAP1). A GAP1 strain can utilize L-citrulline as the sole nitrogen source but cannot grow in the presence of the toxic amino acid D-histidine. L-citrulline as well as D-histidine uptake is mediated solely by the general amino acid permease, and a gap1 strain is therefore able to grow in the presence of D-histidine but cannot utilize L-citrulline. Gene disruption is effected by transforming a gap1 strain with a gene cassette generated by PCR, containing GAP1 flanked by short (60 bp) stretches of the gene in question. Through homologous recombination, the cassette will integrate into the target gene, which is thus replaced by GAP1, and mutants are selected for on minimal L-citrulline medium. When propagated under non-selective conditions, some cells will lose the GAP1 gene. This is caused by recombination between two Salmonella typhimurium hisG direct repeats embracing GAP1, and will result in a sub-population of gap1 cells. Such cells are selected on a medium containing D-histidine, and may subsequently be used for a second gene disruption, Hence, multiple gene disruptions can be made fast, cheaply and easily in a gap1 strain, with two positive selection steps for each disruption. Copyright (C) 2000 John Wiley and Sons, Ltd.