SummaryThenit‐4genes of three conventionalNeurospora crassamutations and of the closely related species,Neurospora intermedia, have been isolated by amplifying the genomic DNA with the polymerase chain reaction. Nucleotide sequencing has revealed that the threenit‐4mutants, alleles 15,1214, and 2994, are the result of a missense mutation, a nonsense mutation and a frameshift mutation, respectively. The nucleotide sequence of the NIT4 protein coding region of anit‐4mutant (allele 2994) and ofN. intermediahave been determined and compared with that of wild‐typeN. crassa.The molecular characteristics confirm that the mutated gene of 2994 originated fromN. intermediaand was introgressed intoN. crassa.The polyglutamine domains of theN. crassawild type, the 2994 mutant, orN. intermediacannot replace an upstream glutamine‐rich domain which is essential fornit