All tested proteolytic Clostridium botulinum type A, B, and F strains and certain non-proteolytic B and F cultures produced a protease having trypsin-like substrate specificity; none of the tested type E (non-proteolytic) strains produced the enzyme. Progenitor toxin (toxic form whose specific toxicity is increased by treatment with trypsin) was found in culture fluid concentrates of all strains not producing the protease; it was also present in some concentrates that had the enzyme. Activation of highly purified type E progenitor toxin (molecular weight 150,000) by essentially pure protease from a proteolytic type B culture was always less than that obtained with trypsin. The product of the type E progenitor toxin-protease reaction increased in toxicity when further treated with trypsin. Results suggest that at least two bonds are cleaved by trypsin during activation of type E progenitor toxin to toxin (form manifesting maximal possible specific toxicity). Natural activation of progenitor toxin of proteolytic strains may also involve cleavage of more than one bond.