Liposomes of [3H]sphingomyelin are readily hydrolyzed by extracts of human spleen, liver, cultured skin fibroblasts and purified placental sphingomyelinase in the absence of detergents. The pH optimum for hydrolysis by liver and spleen extracts was 6.5-7.0 while the fibroblast activity showed an optimum at pH 4.0-4.3. However, the pH optimum for purified placental sphingomyelinase in the presence of Triton X-100 (pH 5.0) is only slightly different from that displayed with liposomes (pH 5.3). The data clearly show that hydrolysis of liposomal sphingomyelin by sphingomyelinase is affected by the composition and purity of the enzyme source.