Sphingosine has been shown to modulate neurotransmitter release. Because membrane fusion and fission involve lipid metabolism, we asked here whether sphingosine had a role in regulating endocytosis. To explore this hypothesis, we monitored changes of membrane capacitance (Cm) to study the effects of intracellular sphingosine on membrane retrieval after chromaffin cell stimulation with depolarising pulses (DPs). We found that: (1) sphingosine dialysis through the patch-clamp pipette (SpD) using the whole-cell configuration of the patch-clamp technique (WCC) favours the appearance of a pronounced endocytotic response; (2) SpD-elicited endocytosis was Ca(2+)-dependent but Ba(2+) did not substitute Ca(2+); (3) under WCC, such endocytotic response disappeared with repetitive DPs; (4) in cells preincubated with sphingomyelinase to augment endogenous sphingosine synthesis, and then voltage-clamped under the perforated-patch configuration of the patch-clamp technique (PPC), endocytosis decayed little with repeated stimulation; (5) sphingosine-1-phosphate (S1P), a metabolite of sphingosine, had a meagre effect on endocytosis; and (6) neither dynamin inhibitor dynasore nor calmodulin blocker calmidazolium affected the sphingosine elicited endocytosis. We believe this is the first report showing that sphingosine plays a permissive role in activating Ca(2+)-dependent endocytosis during cell depolarisation. This effect requires high subplasmalemmal cytosolic Ca(2+) concentrations and a cytosolic factor(s) that is dialysed with the pipette solution. Independence of dynamin and calmodulin suggests that sphingosine-dependent endocytosis could be a novel, more direct pathway for vesicle recycling under mild depolarisation stimuli.