Sphingosine-1-phosphate (SPP) has been proposed to act both as an intracellular second messenger and as an extracellular mediator via specific cell surface receptors. Based on the increasing diverse cellular roles methods to quantify endogenous and exogenous SPP are highly required. Here, we report a rapid HPLC method that allows quantification of SPP in the picomolar range even in complex biological systems. A two-step lipid extraction serves to separate SPP from most interfering phospholipids and sphingolipids. Importantly, dihydrosphingosine-1-phosphate (dihydro-SPP), not detectable in all cultured cells and biological samples in considerable amounts, possesses equal extraction properties and therefore is an ideal internal standard. Following extraction SPP and dihydro-SPP are converted to fluorescent isoindol derivatives by ortho-phthaldialdehyde (OPA) and separated by HPLC using a gradient program with methanol and 0.07 M K2HPO4 as eluents. With this procedure we were able to obtain reproducible measurements of SPP over a broad range from 0.5 pM to 0.2 nM. The identity of SPP and dihydro-SPP was confirmed by the use of the ion pair reagent tetraammoniumsulfate, which induced a shift of both peaks but did not alter peak areas. Moreover, enzymatic conversions to sphingosine and sphinganine by bovine intestinal mucosa alkaline phosphatase (AP) excluded the existence of overlapping compounds. Levels of SPP were determined in a variety of biological samples like serum, thrombocytes, primary keratinocytes and several cell lines. Furthermore, we were able to detect increases of intracellular SPP levels in human keratinocytes after exposure to 1alpha,25-dihydroxyvitamin D3(1,25-(OH)2D3) for which a stimulation of sphingosine kinase activity has been recognized.