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SUMMARY: In three independently obtained mutant strains of Aspergillus nidulans, resistant in different degrees to acriflavine, resistance is due, in each case, to mutation in a single gene. Two of the mutant alleles, ACR1 and ACR3, are semi-dominant and either allele confers a high degree of resistance. These alleles are located about 23 units distal to the w (white conidia) locus and are presumably allelic. A cross involving these two alleles in repulsion gave 0.1% sensitives. A third mutant allele (acr2) is also located on the w chromosome, but on the other arm about 25 units distal to the ad1 locus and over 100 units distant from the ACR1 and ACR3 loci. This allele, which confers relatively slight resistance, is almost completely recessive. Diploid strains which carry any allele for resistance in heterozygous condition give, by vegetative segregation, haploid and homozygous diploid resistant types which are preferentially selected on medium with acriflavine. The use of this technique for the automatic selection of vegetative segregants provides an additional tool for analyses through the parasexual cycle.