To gain information on the long-term effects of cigarette mainstream smoke (MS) on the progression of atherosclerosis, male Apolipoprotein A deficient (ApoE-/-) mice (20/group) were whole-body exposed for 12 months (6 hours/day, 5 days/week) to diluted MS at total particulate matter (TPM) concentrations of 100 μg/l and 200 μg/l or to filtered fresh air (sham). To further investigate the effect of MS in combination with elevated fat levels, mice were fed either a chow diet (0.02% cholesterol / 4.5% fat) or a high-fat diet (0.17% cholesterol / 21% milk fat). Intimal area covered by plaque in the aortic arch, cholesterol content in the aortic arch, and plaque size in the brachiocephalic artery (BA) were higher in the high-fat diet groups compared to the chow diet groups (p<0.01 ANOVA). Exposure to 200 μg TPM/L MS in combination with high-fat diet resulted in higher levels of cholesterol in the aortic arch and increased plaque size in the BCA as measured over the entire time course compared to sham (p<0.05 linear regression), with a 59% (0.13 ± 0.02 vs 0.2 ± 0.01 mm2) increase in plaque size at 6 months (p<0.05 ANOVA). Plaque size in the BA was also significantly higher in chow-fed mice exposed to 200 μg TPM/L MS as measured over the entire time course compared to sham (p<0.05 linear regression), with an 86% (0.05 ± 0.01 vs 0.09 ± 0.03 mm2) enhancement at 6 months (not statistically significant). In the high-fat diet groups, exposure to 100 μg TPM/L MS and to 200 μg TPM/L MS significantly altered the number of elastin positive layers, indicating changes in plaque morphology, at both 6 and 9 months (p<0.01 ANOVA). These data suggest that long-term exposure to cigarette mainstream smoke accelerates the development of atherosclerosis in Apo E-/- mice, particularly in combination with a milk-fat-enriched diet.