Lipoprotein-X containing plasma from a patient with familial lecithin:cholesterol acyltransferase (LCAT) deficiency, was used as substrate and incubated with postheparin plasma or partly purified lipases. LP-X could not be demonstrated by agar gel electrophoresis after incubation with postheparin plasma from a healthy subject, from a patient with chronic active hepatitis deficient in hepatic lipase, or with partly purified lipoprotein lipase. After incubation a marked increase in free fatty acids (FFA) was observed. In contrast LP-X was still present after incubation when postheparin plasma deficient in lipoprotein lipase or partly purified hepatic lipase was added to the substrate. Only minor changes in the concentration of FFA occurred. After addition of oleic acid to the substrate LP-X could not be demonstrated by agar gel electrophoresis. However, in the isolated low density lipoproteins, LP-X like particles were still present as viewed by electron microscopy. Our results strongly suggest that the change in electrophoretic mobility of LP-X was induced by the release of FFA. This was achieved by lipoprotein lipase, but not by hepatic lipase.