A phospholipase has been purified about 50-fold from rat and calf brain, using separae procedures for each of these sources. The enzyme from both tissues hydrolyzed the 1 (or α′) position of lecithin but not the 2 (or β) position of this substrate. It is therefore classified as a phospholipase A1. In the presence of Triton X-100 the enzyme from rat brain hydrolyzed phosphatidyl ethanolamine 5 times slower and that from calf brain, 3 times slower than lecithin. If both Triton X-100 and sodium taurocholate were added to the reaction mixtures, phosphatidylethanolamine and phosphatidylcholine were hydrolyzed at about the same rates. Both preparations hydrolyzed lysolecithin at only 2-5% of the corresponding rate of hydrolysis of lecithin. The rat brain phospholipase had very low lipase activity, while the enzyme from calf brain hydrolyzed tripalmitin at about two thirds of the rate of hydrolysis of lecithin. Phospholipase activity was stimulated by the addition of the nonionic detergent, Triton X-100, or the anionic detergent, sodium taurocholate, calcium ions had no effect on the reaction. The pH optimum of the reaction was 4.0 and the Km 0.8 mM. The reaction was competitively inhibited by fatty acid; the Ki, using palmitate, was 0.07 mM, i.e. 11 times lower than the Km.