Sphingoid bases are precursors and breakdown products of sphingolipids and may function as second messengers. Here we have tested the hypothesis that sphingoid bases are produced in cells in response to external stimuli. Using a high-performance liquid chromatography system, the pattern and the amounts of free sphingoid bases in various cell types (i.e., NIH-3T3, A431, NG108-15) were determined. The predominant sphingoid base in these mammalian cells was identified as C-18 sphingosine, followed by C-18 sphinganine (dihydrosphingosine). In all cells examined, the levels of endogenous sphingoid bases can be rapidly elevated by replacing cell-conditioned medium with Hepes-buffered saline or with fresh medium, causing a dramatic increase (up to 9.5-fold) in sphingosine levels within 60 min; sphinganine levels were raised to a lesser extent (up to 4.5-fold). Addition of ammonium ions inhibited the generation of sphingoid bases. These results suggest that the machinery for metabolizing sphingoid bases can be stimulated rapidly, although the exact nature of the stimulus remains obscure. Nevertheless, the ability to control sphingosine formation in cells by changing medium conditions provides a powerful tool for investigations of the physiological roles of endogenous sphingosine.