We describe here in detail the development of a method to quantitatively measure sphingosine 1-phosphate (Sph-1-P), a bioactive sphingolipid. Sph-1-P was first extracted from cells into the upper aqueous phase under alkaline conditions by Folch's phase separation and then reextracted into the lower chloroform phase under acidic conditions. This phosphorylated sphingoid base extracted was quantitatively converted to N-[3H]-acetylated Sph-1-P, that is [3H]C2-ceramide 1-phosphate (C2-Cer-1-P), by N-acylation with [3H]acetic anhydride. The [3H]C2-Cer-1-P formed with the acylation was resolved by thin-layer chromatography, detected with autoradiography, and quantitated by scraping the corresponding band and counting its radioactivity with a scintillation counter. This assay allows quantification of Sph-1-P over a range from at least 100 pmol (often 30 pmol) to 10 nmol (the highest level tested). The utility and validity of our assay were demonstrated using human platelets. The amount of Sph-1-P in platelet extracts was proportional to the cell number and calculated as 141 +/- 4 pmol/10(8) cells (mean +/- SD, n = 3), which was about four times higher than that of sphingosine. The potent agonist thrombin did not affect the total Sph-1-P amounts in platelet suspensions but induced the release of Sph-1-P stored in the cells into the medium.