Protoplasts of Choanephora cucurbitarum were obtained by treatment of germlings with commercial enzymes. Colonies originated from regenerated protoplasts showed the same mating type and colony type as their respective parent. Colonies from fused protoplasts of two (+) isolates were of the (+) type, while those from two (−) isolates were of the (−) type. Colonies derived from fused protoplasts of (+) and (−) isolates were self-fertile and produced azygospores and zygospores subsequently. This result supports the hypothesis that azygospores of C. cucurbitarum are single-suspensor zygospores. Mating type heterokaryons resulting from fused protoplasts of (+) and (−) isolates with different colony types segregated into component isolates indicating that karyogamy and meiosis did not occur.