A simple and sensitive method for quantification of sphingosine in cellular lipid extracts was developed. The assay is based on quantitative conversion of sphingosine to (\mathrm{N}-\left[{ }^{3} \mathrm{H}\right]) acetylated sphingosine (\left[\left[{ }^{3} \mathrm{H}\right] \mathrm{C}{2}{ }^{-}\right.) ceramide) by (\mathrm{N})-acylation with (\left[{ }^{3} \mathrm{H}\right]) acetic anhydride under certain conditions. Sphingosine was extracted from cultured cells with chloroform and methanol and then treated with base to remove interfering glycerolipids having reactive amino groups (e.g., phosphatidylethanolamine or phosphatidylserine). Sphingosine was acylated with (\left[{ }^{3} \mathrm{H}\right]) acetic anhydride in the presence of (0.004 \mathrm{~N} \mathrm{NaOH}). Acylation was complete in (1 \mathrm{~h}) at (37^{\circ} \mathrm{C}) when sphingosine was present in the picomole range. After the acylation, samples were treated with (\mathrm{NaOH}) to reduce background radioactivity by removing the remaining (\left[{ }^{3} \mathrm{H}\right]) acetic anhydride and hydrolyzing any ester linkages formed during the acylation and resolved by thin-layer chromatography. (\left[{ }^{3} \mathrm{H}\right] \mathrm{C}{2})-ceramide converted from sphingosine with the acylation was detected with radioautography and quantitated by scraping the corresponding band and counting its radioactivity with a scintillation counter. (\left[{ }^{3} \mathbf{H}\right] \mathrm{C}_{2})-ceramide formed was quantitatively measured. This assay allows quantification of sphingosine over a range of 10 to (1500 \mathrm{pmol}). The amount of sphingosine in lipid extracts from cultured cells was proportional to the number of cells. Sphingosine levels in human gastric cancer KATO III cells, human promyelocytic leukemic HL60 cells, and human monoblastic (\mathbf{U 9 3 7}) cells, determined by this method, were (26.6 \pm 2.2,6.3 \pm 0.4), and (6.8 \pm) 0.6 pmol per (10^{6}) cells, respectively. Our new procedure allows quantification of sphingosine levels present in the low picomole range in lipid extracts from biological samples. (โฟธ 1994) Academic Press, Inc.