The study objectives were to confirm the incidence of cholestasis and determine factors that contribute to its development and the natural course of cholestasis in neonates treated with extracorporeal membrane oxygenation (ECMO). This was a retrospective chart review including all patients receiving ECMO between 1995 and 2005 at Kosair Children's Hospital. Neonates were grouped as having cholestasis or no cholestasis. A total of 211 patients underwent ECMO between 1995 and 2005. Thirty patients (14%) developed cholestasis. There was no difference in demographics or diagnoses between patients developing cholestasis on ECMO vs. those who did not. The development of cholestasis on ECMO was related to run time, 148 hours +/- 111 (no cholestasis group) vs. 252.8 hours +/- 187 (cholestasis group), p < 0.001 respectively. The majority of patients received veno-arterial ECMO, but there was no difference in cholestasis related to type of ECMO support. Direct hyperbilirubinemia and elevation of hepatic enzymes resolved in all but one patient by the time of discharge. Our incidence of cholestasis (14%) on ECMO is lower than previously reported series. Longer duration of ECMO and other complications on ECMO (renal, infectious, and metabolic) are more likely in the patients developing cholestasis. Follow-up showed resolution of cholestasis in all patients without hepatic sequelae.