Agaricus bisporus , the cultivated button mushroom, has a mostly secondarily homothallic life cycle. This mode of sexual reproduction could limit outcrossing and recombination among homokaryons in natural populations and also creates difficulties in mushroom breeding. An alternative source of recombinant genotypes is from somatic pairings of heterokaryons. In this study, two homokaryon × heterokaryon and three heterokaryon × heterokaryon pairings were made to examine the possibility of somatic recombination. Four subcultures from the confrontation zone of each pairing were taken for analysis of restriction fragment length polymorphisms at 18 nuclear loci representing seven linkage groups and two regions of mitochondrial DNA. A strikingly high frequency of somatic recombination was observed. Five of the eight subcultures from the two homokaryon × heterokaryon pairings and five of the 12 subcultures from the three heterokaryon × heterokaryon pairings were recombinant. No loss of marker alleles was detected in any of the self-self pairings of the six original strains. No recombination was observed between the two regions of mtDNA examined in this study. The recombination among nuclear loci involves nuclear reassortment, exchange of genetic material between nuclei, and, in one case, crossing over between markers located on the same chromosome. While the mechanism of somatic recombination in A. bisporus is not known, heterokaryons might be used in pairings with other heterokaryons or with homokaryons to produce abundant recombinant genotypes for mushroom breeding.