Tumor necrosis factor-alpha (TNF-alpha) is known to be a secretory product of activated macrophages. TNF-alpha activates endothelial cells, stimulates angiogenesis and induces proliferation of smooth muscle cells (SMCs). Therefore, TNF-alpha has been suggested to be actively involved in the inflammatory events associated with atherosclerosis. Previous ultrastructural and immunocytochemical studies have shown that macrophages as well as SMCs are constituents of atherosclerotic lesions in the Watanabe heritable hyperlipidemic (WHHL) rabbit. Recently, we have shown that TNF-alpha mRNA levels in aorta of 18-month-old WHHL rabbits were significantly higher than that of 6-month-old WHHL rabbits and New Zealand White (NZW) rabbits by quantitative RT-PCR . However, it remains unclear as to the cell type(s) responsible for the increased TNF-alpha mRNA levels in atherosclerotic lesions. In this study, we provided evidence showing the expression of the TNF-alpha gene in the medial SMCs as well as cells of intimal lesions in arteries of WHHL rabbits by in situ transcription (IST). TNF-alpha protein was also detected in the cytoplasm of the intimal and medial SMCs and macrophages by immunocytochemistry using a monoclonal antibody against rabbit TNF-alpha. In contrast, the expression of TNF-alpha mRNA and protein can not be detected in the arteries from healthy New Zealand White (NZW) rabbits. Our results suggest that the expression of TNF-alpha in both intimal and medial SMCs and macrophages is associated with the progression of atherosclerosis.