We previously demonstrated that bacterial sphingomyelinase (SMase) activates a volume-sensitive current, ICl,swell, by a pathway that involves mitochondrial ROS production. SMase activity generates endogenous ceramides from sphingomyelin in the outer plasma membrane leaflet and, in turn, ceramides are metabolized to several sphingolipids, including sphingosine-1-phosphate (S1P). We tested whether ceramide metabolites are responsible for eliciting ICl,swell. Under isosmotic conditions that isolate anion currents, SMase-induced ICl,swell was abrogated by blockade of ceramidase (converts ceramide to sphingosine) with D-erythro-MAPP (10 μM). SMase-induced ICl,swell also was suppressed by inhibition of sphingosine kinase with DL-threo-dihydrosphingosine (10 μM). These data suggested that the ceramide metabolite S1P is likely to stimulate ICl,swell. As expected, exogenous S1P (500 nM) elicited an outwardly rectifying Cl- current that was fully inhibited by the ICl,swell-specific blocker DCPIB (10 μM). As seen with SMase-induced ICl,swell, S1P-induced ICl,swell was fully inhibited by the mitochondrial Complex I blocker rotenone (10 μM), which suppresses extramitochondrial ROS release by Complex III. In contrast to results with SMase, S1P-induced current was partially inhibited by blockade of NADPH oxidase (NOX) with apocynin (500 μM). These data indicate that S1P is a necessary component of SMase-induced ICl,swell activation and that the action of exogenous S1P involves ROS from both mitochondria and NOX. Importantly, exogenous C2-ceramide (2 μM), a synthetic short-chain ceramide, also elicits ICl,swell even though C2-ceramide is not metabolized to S1P in native cells. Thus, it seems likely that ceramides can elicit ICl,swell via S1P and also by a distinct pathway and that both pathways converge at mitochondrial ROS.