An A alpha mating-type allele (A alpha 4) was isolated by walking the chromosome from the closely linked PAB1 gene. A cosmid clone containing the A alpha 4 allele isolated from the walk was used as a probe to recover the A alpha 1 allele from another cosmid library. Cosmids encoding mating-type activity were identified by transforming Schizophyllum cells and screening for activation of A-regulated development. Putative mating-type transformants were confirmed in mating tests and genetic analyses of progeny. The identity of the specific alleles isolated was demonstrated by showing that their effectiveness in transforming for mating type is limited to recipient strains possessing an A alpha allele different from the one encoded by the cloned sequences. Transforming DNA is active in trans, suggesting that A alpha encodes a diffusible product. Restriction mapping shows that A alpha 1 and A alpha 4 are coded in the same physical region of the genome, but within a subregion that contains extensive sequence divergence. In addition, Southern analyses show that there is only one copy of A alpha 1 or A alpha 4 per haploid genome, and that they do not cross-hybridize to one another or to any of the other A alpha alleles. A alpha 1 and A alpha 4 were subcloned as 2.8 and 1.2 kb fragments, respectively, retaining in transformation all the mating-type activity demonstrated of the original cosmids.