C12 NBD ceramide (d18:1/12:0) is a long-chain fluorescent ceramide analog that can be used for the measurement of alkaline and neutral ceramidase activity.{14063} It is hydrolyzed efficiently by alkaline ceramidase from P. aeruginosa and by neutral ceramidase from mouse liver. In addition, alkaline and neutral ceramidases from a variety of mammalian cell lines also hydrolyze C12 NBD ceramide (d18:1/12:0). Acid ceramidase does not appear to utilize C12 NBD ceramide (d18:1/12:0) as a substrate. The possibility exists forC12 NBD ceramide (d18:1/12:0) to also be applied to the measurement of ceramide kinase and/or sphingomyelin synthase activity.