Mouse aortic smooth muscle cells (SMCs) were loaded for 72 h with cholesterol by using cholesterol:methyl-β-cyclodextrin complexes, leading to ≈2-fold and ≈10-fold increases in the contents of total cholesterol and cholesteryl ester, respectively. Foam-cell formation was demonstrated by accumulation of intracellular, Oil Red O-stained lipid droplets. Immunostaining showed decreased protein levels of smooth muscle α-actin and α-tropomyosin and increased levels of macrophage markers CD68 and Mac-2 antigen. Quantitative real-time RT-PCR revealed that after cholesterol loading, the expression of SMC-related genes α-actin, α-tropomyosin, myosin heavy chain, and calponin H1 decreased (to 11.5 ± 0.5%, 29.3 ± 1.4%, 23.8 ± 1.4%, and 3.8 ± 0.5% of unloaded cells, respectively; P < 0.05 for all), whereas expression of macrophage-related genes CD68, Mac-2, and ABCA1 mRNA increased (to 709 ± 84%, 330 ± 11%, and 207 ± 13% of unloaded cells, respectively; P < 0.05 for all), thereby demonstrating that the protein changes were regulated at the mRNA level. Furthermore, these changes were accompanied by a gain in macrophage-like function as assessed by phagocytotic activity. Expression of vascular cell adhesion molecule 1 and monocyte chemoattractant protein 1, known responders to inflammation, were not changed. In conclusion, cholesterol loading of SMC causes phenotypic changes regulated at the mRNA level that result in a transdifferentiation to a macrophage-like state. This finding suggests that not all foam cells in lesions may have a macrophage origin, despite what is indicated by immunostaining for macrophage-related markers. Furthermore, inflammatory changes in foam cells observed in vivo may not be simple consequences of cholesterol accumulation.