An isolate of Metarrhizium anisopliae from the eastern United States was found to produce substances which caused tetanic paralysis in larvae of the greater wax moth, Galleria mellonella, when injected into the hemocoel. The production of these materials in vitro was investigated and it was found that they were present in greater amounts in submerged cultures than in surface cultures, even when dry mycelial weights were approximately equal. The toxins were produced with either inorganic or organic nitrogen as the nitrogen source in the culture medium. They were released by growing colonies and titers did not increase as the colonies lysed, which indicates they were exotoxins.

A bioassay system was devised for determining nonlethal titers of toxins. The criterion for positive response was inability of the test larva (Galleria mellonella) to right itself when placed on its back 5 minutes after injection. ED50's were estimated by plotting normit of response against logarithm of dose (in microliters per gram larval weight).