Pairings of single-spore isolates from different basidiocarps were made to examine the numbers and distributions of incompatibility factors in the tetrapolar basidiomycetes Pleurotus ostreatus and Pleurotus sapidus. These factors do not appear to be randomly distributed, with multiple basidiocarps on a single tree or log arising from a single mycelium, multiple basidiocarps on a single tree or log sharing a single common monokaryotic parent, and a tendency toward spatial clustering of factors even when multiple basidiocarps from the same tree or log are excluded from the analyses. For one 30-km2 collection area, the estimated numbers of A and B factors in the P. ostreatus population were 126 and 354, respectively, yielding an estimated outbreeding efficiency of 0.99. Estimated outbreeding efficiencies were quite high [Formula: see text] even in a sample taken from a very small area (0.09 m2), illustrating the ability of the tetrapolar mating system to simultaneously prevent self mating and minimize the number of incompatible matings. Key words: Basidiomycotina, fungi, mating system, population genetics.