1. A simple method is given for isolating from ram spermatozoa a water-soluble form of acrosin (a trypsin-like enzyme) which is about 25% pure. It is free from an acrosin inhibitor which is located in the spermatozoa. 2. In the hydrolysis of N-α-benzoyl-l-arginine ethyl ester the degree of activation of acrosin by Ca²⁺, and by some other cations, is dependent on the extent of contamination by the inhibitor. In 50mm-Tris–HCl buffer (pH8.2) activation by Ca²⁺ did not exceed 40%, but acrosin that is partially inhibited may be activated by up to 300%: this is due to cation-mediated protection of acrosin against the inhibitor. 3. Increasing concentrations of buffers (e.g. Tris) also activate acrosin but at above certain buffer concentrations Ca²⁺ no longer exerts an activating effect and may become inhibitory. Ca²⁺ is also inhibitory when added to assay systems involving anionic buffers with chelating properties. This is due to a fall in pH. 4. The above results suggest reasons for conflicting conclusions in papers dealing with the effects of Ca²⁺ on acrosin activity. 5. Inhibition of acrosin by the Kunitz pancreatic trypsin inhibitor is increased on addition of Ca²⁺. Inhibitions of trypsin by the acrosin inhibitor and by the Kunitz inhibitor are insensitive to Ca²⁺. 6. Like trypsin, acrosin is activated, up to 60%, by 2-methyl-propan-2-ol, dimethyl sulphoxide, and some other water-miscible solvents. Effects of cations and solvents tend to be additive and a common maximum acrosin activity can be achieved with various concentrations of solvent, salts and buffer in the assay system. Activation by solvents is increased when low concentrations of the acrosin inhibitor are present. 7. Activations of acrosin by salts and by solvents are more pronounced when the substrate is N-α-benzoyl-dl-arginine 2-naphthylamide. 8. K_m values for ram acrosin (about 0.2mm) are much higher than those for trypsin, and k_cat. values are slightly higher than those for trypsin. Considerations of the influences of ions and dimethyl sulphoxide on the activities and kinetic constants of acrosin and trypsin suggest that conformational changes are the factors mainly responsible for the reported activations of acrosin. 9. The following conclusions are reached. (a) Acrosin plays a role in the penetration of the sperm cell into the egg without becoming detached from the acrosomal membrane. (b) The enzyme is a peripheral membrane protein which may be classed as a cathepsin. (c) The susceptibility of the activity of soluble acrosin to cations and solvents points to a flexible molecule, i.e. one lacking conformational restraints imposed by association (presumably ionic) with the acrosomal membrane.

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