Cathepsin D was purified from porcine spleen to near homogeneity as determined by gel electrophoresis. The isolation scheme involved an acid precipitation of tissue extract, DEAE-cellulose and Sephadex G-200 chromatography, and isoelectric focusing. The end product represented about a 1000-fold purification and about a 10% recovery. The purified enzyme was the major isoenzyme, which represented 60% of cathepsin D present in porcine spleen. Two minor isoenzymes of cathepsin D were present in small amounts. The purified enzyme resembled porcine pepsin in molecular weight (35,000), amino acid composition, and inactivation by specific pepsin inactivators. The pH activity curve of the purified enzyme showed two optima near pH 3 and 4. The relative activities at these optimal pH values were affected by salt concentration. Experimental evidence indicated that the two-optima phenomenon is a property of a single enzyme species.