Inbreeding depression was observed in the commercial button mushroom, Agaricus bisporus, by examining two laboratory populations. The outbred population consisted of 20 compatible pairings, 10 homokaryons with each of the homokaryons Ag1-1 and Ag89-65. The inbred population consisted of 104 backcrosses (among which 52 were expected to be sexually compatible) obtained from the pairings of two progenitor homokaryons, Ag1-1 and Ag89-65, with 52 progeny homokaryons derived from the mating between Ag1-1 and Ag89-65. The eight fitness components examined for these two populations were successful matings as identified by the analysis of restriction fragment length polymorphisms, positive mycelial interaction in these successful matings, heterokaryon growth rate, primordium formation by the successful matings, fertile fruiting body formation, time to first break, average number of fruiting bodies per square foot, and average weight per fruiting body. The outcrossed population showed a significant advantage over the inbred population in three of eight fitness components. Two pairs of traits were significantly correlated. The multiplicative fitness ratio of the inbred to the outcrossed population was 0.18. The relevance of inbreeding depression to the evolution of fungal mating systems and to mushroom breeding is discussed.