A molecular investigation was conducted on the production of type II ribotoxin of the species Aspergillus and related fungi in Taiwan. Species that carried ribotoxin were confirmed by (1) cross-reactivity to anti-alpha-sarcin serum; (2) Southern dot hybridization; (3) PCR amplification of genomic DNA with specific primers; and (4) analysis of ribotoxic activity. Five new strains, A. clavatus, A. oryzae var. effusus, A. ostianus, A. tamarii, and Neosartorya fischeri var. spinosa, were identified to contain an alpha-sarcin-like ribotoxin. These positive strains exhibit ribotoxic activity by cleaving ribosomes and generating an alpha-fragment.