Sphingomyelin is an abundant constituent of the plasma membranes of mammalian cells. Ceramide, its primary catabolic intermediate, is released by either acid sphingomyelinase or neutral sphingomyelinase (nSMase) and has emerged as a potential lipid signaling molecule. nSMase is regarded as a key enzyme in the regulated activation of the "sphingomyelin cycle" and cell signaling. We report here the cloning, identification, and functional characterization of murine and human nSMase, a ubiquitously expressed integral membrane protein, which displays all established properties of the Mg2+-dependent nSMase of the plasma membrane. Stably nSMase-overexpressing U937 and human embryonic kidney cell lines have been generated for the study of the role of nSMase in signal transduction pathways. Their stimulation by tumor necrosis factor alpha leads only to a moderately elevated ceramide concentration. Activation of Jun kinase and NFkappaB and poly(ADP-ribose) polymerase cleavage are identical in mock- and nSMase-transfected cells. Tumor necrosis factor alpha triggers the ERK1 pathway in none of the cell lines. The cloned nSMase will facilitate further controlled experiments aiming at the definition of a possible role of ceramide as signal transduction molecule.