Sphingosine kinase (SPHK) phosphorylates sphingosine to form a bioactive lipid mediator, sphingosine 1-phosphate (S1P). S1P mediates such diverse biological processes as regulation of cell differentiation, motility, and apoptosis both extracellularly, via S1P (Edg) family receptors, and intracellularly, through unidentified targets. In cells S1P is short-lived, so the synthetic process catalyzed by sphingosine kinase may be important in maintaining the cellular levels of the compound. Thus far, two sphingosine kinases have been reported, with SPHK1 exhibiting the higher activity. However, several studies suggest the existence of unidentified sphingosine kinases. Therefore, to further elucidate the role of SPHK1 in the formation of S1P, we investigated its contribution to the total sphingosine kinase activity in mouse tissues. We found that SPHK1 is a major sphingosine kinase in many tissues, especially in brain, heart, and colon. However, some tissues such as spleen, small intestine, and lung contained sphingosine kinase activity that was not attributable to SPHK1 or SPHK2, as determined by immunodepletion assays. Furthermore, the presence of other sphingosine kinases with different properties, i.e., higher activity toward phytosphingosine and a different subcellular distribution, is suggested.