Fractalkine is distinguished structurally from other chemokines in that it contains a mucinlike stalk that tethers a CX3C chemokine module to a transmembrane‐spanning region; its expression in cultured endothelial cells has been shown to be up‐regulated by tumor necrosis factor α (TNF‐α) and interleukin‐1 (IL‐1). The purpose of this study was to determine whether fractalkine is expressed, in a proinflammatory agent‐regulated manner, by cardiac endothelial cells in vivo. Steady state levels of fractalkine mRNA were increased in rat cardiac tissues after in vivo treatment with lipopolysaccharide (LPS), IL‐1, or TNF‐α. In situ hybridization and immunohistochemical analysis revealed that endothelial cells of the coronary vasculature and endocardium were the principal source of proinflammatory agent‐inducible fractalkine, although some fractalkine immunoreactivity was also found on the myocytes. These data are the first demonstration of in vivo cardiac endothelial cell fractalkine expression and regulation by proinflammatory agents such as LPS, IL‐1, or TNF‐α. Cardiac endothelial cell‐expressed fractalkine may contribute to the influx of leukocytes into the heart during inflammation. J. Leukoc. Biol. 66: 937–944; 1999.