<h4>Objective</h4>Recent studies have indicated that plasma sphingomyelin levels and sphingomyelinase activity are risk factors for atherosclerosis. Therefore, it is suggested that ceramides, which are hydrolyzed products of sphingomyelin and a biologically active lipid causing apoptosis in a variety of cells, have an important role in the incidence of atherosclerosis. In this study, we examined whether cholesterol- and fat-enriched diets, which are causes of atherosclerosis, affect ceramide metabolism. In addition, we found a relation among lipid markers of atherosclerosis such as cholesterol, triacylglycerol, and ceramide concentrations.<h4>Methods</h4>Male Wistar rats were fed a diet supplemented with 1% cholesterol or 30% high-fat diet for 8 wk. Tissue ceramide levels were analyzed using electrospray tandem mass spectrometry.<h4>Results</h4>The major ceramides in plasma and the liver were C24:0 and C24:1. The major ceramides in adipose tissues were C16:0 and C24:0. Therefore, the ceramide composition of the adipose tissues was different from that of plasma and the liver. In addition, total ceramide levels in plasma and the adipose tissues of rats fed cholesterol were higher than those in the control group.<h4>Conclusion</h4>The accumulation of cholesterol caused an increase in ceramides, which might be a new risk factor for atherosclerosis.