Axoplasm extruded from the giant axon of the squid contains Ca2+-activated proteases. The protease in the 100,000 x g of supernatant of axoplasm is very specific and degrades only the 200,000 MW, neurofilament protein (NF200), whereas the protease(s) in the pellet has a much wider range of substrate specificity. The activation of the supernatant protease is restricted to the Ca2+ ion, and no other divalent cation will substitute. The protease requires Ca2+ at a higher concentration than 0.5 mM for activation, and has a pH optimum of about 7.5. Degradation of the NF200 appears to proceed through a 100,000 MW and possibly a 47,000--50,000-MW intermediate form before degradation to TCA-soluble peptides. Activity of the protease is inhibited by divalent cation chelators, Cu2+ and Fe2+, sulphydryl inhibitors, and leupeptin. This specific Ca2+-activated protease in squid axoplasm has identical properties to Ca2+-activated proteases found in various non-neural tissues. Despite its narrow protein substrate specificity, Ca2+-activated protease purified from human platelets effectively degrades squid NF200, suggesting a possible structural relationship between platelet and muscle actin-binding proteins and neurofilament proteins.