Based on abundant resources of squid as crude material, spermatozoa of adult squid were used selectively. After a serial processes of extraction, hydrolyzation and precipitation, crude product of protamine was yielded. The crude protamine was then puri fied by column chromatograph packed with Sephadex G-25. In this paper such physical and chemical factors as temperature, pH, salt concentration, organic substances, etc that affect antimicrobial activity of protamine were studied. Also, the minimum inhibition concentration of protamine was measured under various conditions.