ABSTRACTDispersal via basidiospores has predictable consequences for fungal population structure. Spatial distribution of 60 heterokaryons in a natural population ofPleurotus ostreatuswas examined through study of their somatic incompatibility interactions and analysis of mating compatibility factors. In order to estimate the minimum genetic relationship detectable by somatic incompatibility testing, pairing studies were performed using sib-composed dikaryons and their parental dikaryons. Most pairings (93–100%) between sib-composed dikaryons ofP. ostreatusresulted in somatic incompatibility responses. Patterns of somatic incompatibility interactions among 60 naturally occurring isolates from a 6-hectare area in the Duke Forest indicate that most isolates represent discrete individuals, with as many as 15 individuals occupying a single log. A total of 53 somatically distinct individuals were identified from the population, distributed among 21 logs. Somatically compatible strains from different logs were not observed. The distribution of mating alleles was analyzed for a sample of 10 dikaryons by analysis of mating interactions between dedikaryotized progeny. The observed patterns of distribution for mating alleles supports the view that somatically compatible isolates in this instance were clones. Isolates arising from different logs were all shown to possess unique mating factors. Although four of seven isolates from a single log each carried unique mating factors, the presence of common mating factors in two remaining pairs of strains also suggests nonrandom mating factor distribution at the level of a single substrate. The results of this study implicate basidiospore dispersal as a primary mechanism for dikaryon establishment. However, further genetic studies are still necessary to determine the extent of different mating patterns between individuals in the population.