We have developed an improved assay method to measure sphingomyelinase activity and to detect this enzyme separated on polyacrylamide gels. The assay of sphingomyelinase activity involved immobilizing [N-methyl-14C]sphingomyelin on polyvinyldiflouride (PVDF) membrane, incubation with sphingomyelinase, and the measurement of radioactivity associated with [14C]phosphocholine. The enzyme activity was dependent on the concentration of sphingomyelin, enzyme, pH, and temperature. Thirty minutes of incubation time was optimal for enzyme activity. This enzyme had a bimodal pH optima, in that optimum enzyme activity was measured at pH 5.4 and 7.4. The detection of sphingomyelinase was pursued by separating the enzyme on a polyacrylamide gel and carrying out the enzyme assay by exposure to [14C]sphingomyelin blotted on a PVDF membrane. The enzyme activity on the PVDF membrane was visualized by autoradiography. A white band (depicting hydrolytic removal of [14C]sphingomyelin from PVDF was observed. Our method of detecting sphingomyelinase by immobilizing sphingomyelin on PVDF membrane may serve as a prototype for assaying various other enzymes in which the hydrolytic product is released into the aqueous phase. Moreover, our method for detecting sphingomyelinase on polyacrylamide gels may be helpful in further studies on the molecular biochemistry of this and related phospholipases.