Mating compatibility reactions were tested using homokaryotic strains belonging to anastomosis group 4 in the Rhizoctonia solani complex. Previous studies on mating patterns employed tuft formation between paired homokaryons as the primary criterion for detection of heterokaryons. In this study, somatic incompatibility reactions and genetic markers were used to confirm the reliability of tufts as an indication for heterokaryotization. Sixteen homokaryotic, single basidiospore strains from 10 different heterokaryotic field isolates of R. solani anastomosis group 4 were paired in all possible combinations on potato dextrose and 1% charcoal agar, and examined for tuft formation and heterokaryotization. From a total of 136 pairings, 25% (7 of 28) of intrastock and 54% (58 of 108) of interstock pairings produced visbile tufts. Putative heterokaryons from aerial tufts developed into colonies with appressed mycelium that were morphologically distinct from and somatically incompatible with both parental homokaryons. Similarly distinct heterokaryons also were isolated from the interaction zone between some paired homokaryons that did not form tufts. Patterns of nuclear migration were examined by sampling explanted hyphae from either side of the hyphal interaction zone. Heterokaryons were detected on one or both sides of the heterokaryotic tuft in about half of these cases, indicating that heterokaryotization was not restricted to the zone corresponding to tuft formation. Randomly amplified polymorphic DNA (RAPD) markers were used to confirm putative heterokaryons. The results from these studies show that tuft formation is not always indicative of heterokaryotization and suggest a limited role for nuclear migration during mating in R. solani.