Whole cell patch-clamp recordings were made to study the regulation of the store-operated calcium release-activated calcium current (ICRAC) by metabolites involved in the sphingomyelin pathway in RBL-2H3 cells. Sphingosine, a regulator of cell growth, inhibits ICRAC completely within 200 s and independently from conversion to either sphingosine 1-phosphate or ceramide. Structural analogs of sphingosine, including N,N-dimethylsphingosine, DL-threo-dihydrosphingosine, and N-acetylsphingosine (C2-ceramide) also block ICRAC. This effect is always accompanied by an elevation of whole cell membrane capacitance. These sphingolipids appear, therefore, to accumulate in the plasma membrane and directly block ICRAC channels. Sphingosylphosphorylcholine also increases capacitance but does not inhibit ICRAC, demonstrating structural specificity and that the elevation of capacitance is necessary but not sufficient for block. Nerve growth factor, which is known to break down sphingomyelin, inhibits ICRAC, and this inhibition can be antagonized by reducing sphingosine production with L-cycloserine, suggesting that ICRAC is a physiologically relevant and direct target of sphingosine. We propose that sphingosine directly blocks ICRAC, suggesting that the sphingomyelin pathway is involved in ICRAC regulation.