Sphingosine-1-phosphate (SPP) has diverse biological functions acting inside cells as a second messenger to regulate proliferation and survival, and extracellularly, as a ligand for G protein-coupled receptors of the endothelial differentiation gene-1 subfamily. Based on sequence homology to murine and human sphingosine kinase-1 (SPHK1), which we recently cloned (Kohama, T., Oliver, A., Edsall, L. , Nagiec, M. M., Dickson, R., and Spiegel, S. (1998) J. Biol. Chem. 273, 23722-23728), we have now cloned a second type of mouse and human sphingosine kinase (mSPHK2 and hSPHK2). mSPHK2 and hSPHK2 encode proteins of 617 and 618 amino acids, respectively, both much larger than SPHK1, and though diverging considerably, both contain the conserved domains found in all SPHK1s. Northern blot analysis revealed that SPHK2 mRNA expression had a strikingly different tissue distribution from that of SPHK1 and appeared later in embryonic development. Expression of SPHK2 in HEK 293 cells resulted in elevated SPP levels. d-erythro-dihydrosphingosine was a better substrate than d-erythro-sphingosine for SPHK2. Surprisingly, d, l-threo-dihydrosphingosine was also phosphorylated by SPHK2. In contrast to the inhibitory effects on SPHK1, high salt concentrations markedly stimulated SPHK2. Triton X-100 inhibited SPHK2 and stimulated SPHK1, whereas phosphatidylserine stimulated both type 1 and type 2 SPHK. Thus, SPHK2 is another member of a growing class of sphingolipid kinases that may have novel functions.