Background - Maternal hypercholesterolemia during pregnancy is associated with markedly enhanced fatty streak formation in human fetal aortas and accelerated progression of atherosclerosis in normocholesterolemic children. Methods and Results - To establish the causal role of maternal hypercholesterolemia in a genetically homogeneous murine model and to test the hypothesis that pathogenic events during fetal development result in persistent changes in arterial gene expression, female LDL receptor-deficient (LDLR-/-) mice were fed regular chow or high-fat diets supplemented with 0.075% or 1.25% cholesterol during pregnancy. Lesion sizes were determined in the aortic origin of their chow-fed offspring at 3 months. Maternal hypercholesterolemia more than doubled lesion sizes in male offspring (P<0.0001 for the 0.0075% cholesterol group). Microarray analysis of the expression of 11 000 murine genes in the nonatherosclerotic descending aorta by Affymetrix gene chips suggested that 139 genes were significantly regulated in offspring of hypercholesterolemic mothers. A subset of 12 of the upregulated transcripts was subjected to secondary analysis by semiquantitative PCR of pooled RNA and 4 genes were found to be upregulated >1.7-fold. Quantitative PCR for one of these genes using RNA from individual mice yielded similar results. Comparative immunostaining for several of the above genes also indicated increased protein content in offspring of hypercholesterolemic mothers. Conclusions - These findings establish an atherogenic effect of maternal hypercholesterolemia in genetically uniform mice and indicate that changes in aortic gene expression persist long after fetal exposure to hypercholesterolemia. In addition to elucidating pathogenic mechanisms initiated during fetal development, this approach may identify genes in morphologically normal arteries that influence the susceptibility to classical risk factors of atherosclerosis.