A proteolytic mutant from Clostridium botulinum type E produced extracellular proteases after the end of exponential growth coinciding with the period of sporulation. Proteases were separated into four fractions by chromatography on a DEAE-cellulose column. One was a sulphydryl-dependent protease that also apparently required a divalent cation for enzyme activity since it was inhibited by EDTA. This enzyme hydrolysed synthetic amide and ester compounds containing an arginine residue, and showed some activity towards L-lysine methyl ester. It appeared that two of the other proteases were serine proteases and the fourth was a metal protease. These last three proteases did not require a thiol agent and did not hydrolyse any of the synthetic amides or esters examined. Only the sulphydryl-dependent protease could activate C. botulinum type B, E and F toxins. The ability of this enzyme to activate type B and E toxins was markedly lower than that of trypsin. The susceptibility of type B toxin to this protease was lower than that of type E toxin. C2 toxin was not activated by this enzyme. It is suggested that the sulphydryl-dependent protease in this proteolytic mutant of C. botulinum type E has properties similar to those of proteases from C. botulinum types B and F.