The cell cycle is controlled by cyclin-dependent protein kinases (CDKs). The activity of these enzymes is directed by inhibitors of CDKs. The 21-kD protein product (P21) of the WAF1/CIP1 gene, which can be transactivated by the protein product of the tumor suppressor gene p53, acts as an inhibitor of cyclin-dependent kinases. To assess whether both P21 and p53 may play a role in the control of cellular proliferation in atherosclerotic lesions, the topographical association between p53, P21, and the proliferation marker MIB1/Ki-67, was analyzed by immunohistochemistry in human carotid atheromatous plaques of 26 patients. p53 immunoreactivity (IR) was present in 26 of 26 cases in the nuclei of virtually all cell types (macrophages [MPs], smooth muscle cells [SMCs], endothelial cells [ECs]) in areas with chronic inflammation in 71.08 +/- 8.28% of the nuclei. p53 staining in the control tissue from human coronary arteries was present in 0.3 +/- 0.45% of the cells (P < .002): P21-IR was present in 24 of 26 specimens in 64.38 +/- 10.13% of the cells (controls: 3.8 +/- 1.85%, P < .002) and localized to nuclei of MPs (CD68 positive) and SMCs (alpha-actin positive), as well as ECs of microvessels present in 21 specimens (21 of 21) and luminal ECs present in 18 specimens (16 of 18). As shown by double labeling, P21-IR colocalized with p53-IR in most MPs (24 of 24), intimal SMCs (22 of 24), ECs of microvessels (19 of 21), and luminal ECs (10 of 16). Interestingly, few p53-positive cells did not show simultaneous P21-IR, and, conversely, not all P21-positive cells demonstrated p53-IR. MIB1/Ki-67-positive cells were identified in 21 of 26 tissue specimens in 3.53 +/- 1.79% of the nuclei (controls: 0%, P < .002) and localized principally to MPs bordering the atheromatous lipid core (21 of 26) and to a few scattered SMCs (16 of 26), ECs of microvessels (13 of 21), and luminal ECs (2 of 18). Most importantly, none of the cells coexpressing P21 and p53 were positive for MIB1/Ki-67-IR, indicating the absence of proliferating activity. In summary, this study demonstrates that P21-IR is present in the atherosclerotic plaque and colocalizes with p53 in most MPs, SMCs, and ECs. The lack of proliferation markers in cells coexpressing p53 and P21 suggests that transcriptional activation of the WAF1/CIP1 gene by p53 may be involved in the control of cellular proliferation in advanced human atherosclerotic plaques.