Recent work has revealed correlations between bacterial or viral infections and atherosclerotic disease. One particular bacterium, Chlamydia pneumoniae, has been observed at high frequency in human atherosclerotic lesions, prompting the hypothesis that infectious agents may be necessary for the initiation or progression of atherosclerosis. To determine if responses to gram-negative bacteria are necessary for atherogenesis, we first bred atherosclerosis-prone apolipoprotein (apo) E(-/)- (deficient) mice with animals incapable of responding to bacterial lipopolysaccharide. Atherogenesis was unaffected in doubly deficient animals. We further tested the role of infectious agents by creating a colony of germ-free apo E(-/)- mice. These animals are free of all microbial agents (bacterial, viral, and fungal). Atherosclerosis in germ-free animals was not measurably different from that in animals raised with ambient levels of microbial challenge. These studies show that infection is not necessary for murine atherosclerosis and that, unlike peptic ulcer, Koch's postulates cannot be fulfilled for any infectious agent in atherosclerosis.