The mating-type locus of Podospora anserina controls fusion of sexual cells as well as subsequent stages of development of the fruiting bodies. The two alleles at the locus are defined by specific DNA regions comprising 3.8 kb for mat+ and 4.7 kb for mat-, which have identical flanking sequences. Here we present the characterization of several mutants that have lost mat(+)-specific sequences. One mutant was obtained fortuitously and the other two were constructed by gene replacement. The mutants are deficient in mating with strains of either mat genotype but are still able to differentiate sexual reproductive structures. The loss of the mating type does not lead to any discernible phenotype during vegetative growth: in particular it does not change the life span of the strain. The mutants can recover mating ability if they are transformed with DNA containing the complete mat+ or mat- information. The transformants behave in crosses as do the reference mat+ or mat- strains, thus indicating that the transgenic mat+ and mat- are fully functional even when they have integrated at ectopic sites.