Ten heterokaryons of Agaricus bisporus (= Agaricus brunnescens) were shown to carry four different mitochondrial (mt) genotypes by analysis of mt restriction fragment length polymorphisms (RFLPs). Fifteen homokaryons derived from these strains were used to investigate mt inheritance in A. bisporus. One hundred eighty-nine pairings were performed in 25 different combinations. Pairings in 15 different combinations produced heterokaryons on the basis of nuclear RFLP analyses and/or fruiting trials. The mt genotype of each new intraspecies hybrid was examined by using mt RFLPs as genetic markers. Our results suggest the following. (i) Recombination between the mt genomes was not a common event. (ii) From most individual pairings, all heterokaryons carried the same mt genotype. (iii) Heterokaryons carrying either of the two possible mt genotypes were observed in certain crosses after modification of the pairing procedure. A biparental transmission pattern was demonstrated for some crosses, but there appears to be a preference for one of the mt genotypes to predominate in any specific pairing.