Interspecific hybridization between Volvariella volvacea and V. bombycina was studied using the protoplast fusion technique. The fusion frequency was found to be in the range of 0.032 to 0.333%. Protoplasts from various hybrids were released and regenerated to determine whether they were heterokaryons. In all regenerated colonies, both parental types could not be recovered at the same time. The nuclear DNA contents of hybrids were compared with their parents, and no diploid (parent 1 genome plus parent 2 genome) was found. Some hybrids revealed novel fragments in mitochondrial rDNA PCR profiles, which indicated that rearrangement of mtDNA could have occurred after fusion. Results from arbitrarily-primed polymerase chain reaction (AP-PCR) fingerprints also revealed that the majority of hybrids were similar to one parental type, but heterologous fragments were found in some hybrids.