A Ca2+-dependent protease was prepared from rat brain by using DEAE-Sephadex, Sephadex G-200, and substrate affinity chromatography. Degradation of neurofilament proteins was determined by measuring the changes in radioactivity of electrophoretically separated bands of radioiodinated neurofilament proteins. The apparent Km values for 68000- (P68), 150000- (P150), and 200000- (P200) dalton neurofilament proteins are 3.9 x 10(-8) M, 4.4 x 10(-8) M, and 8.2 x 10(-8) M, respectively. Proteolytic activity is dependent upon Ca2+ concentration with threshold and saturation values of 10(-6) and 10(-4) M, respectively. The enzyme is also inactivated by preincubation with Ca2+. Similar Ca2+ concentrations cause activation and inactivation of enzyme, but the process of inactivation is intrinsically slower than the process of activation. The enzyme is sensitive to thiol protease inhibitors, is activated by Sr2+, Ba2+, Mn2+, and La3+ at 1-10 mM, and has an optimal pH range of 7.4-8.0.