Expression of cytokeratins (CK) is considered a hallmark of the state of epithelial differentiation. CK also occur in certain vascular smooth muscle cells (VSMC), inferring an association with a less differentiated phenotype. Recently, CK posttranslational modification was shown to occur in epithelial cells in stress, mitosis or apoptosis. The aim of this study was to determine potential CK phosphorylation patterns in human VSMC. Tissue samples of normal peripheral and coronary arteries, atherosclerotic lesions and umbilical cord vessels were evaluated by immunofluorescence microscopy applying antibodies specific for cytokeratins 8 and 18, specific cytokeratin phosphorylation sites, Ki-67-antigen as a proliferation marker and nick end labeling (TUNEL) to detect apoptosis. All samples contained cytokeratin-positive VSMC but diverse phosphorylation patterns. The C-terminal serine 431 of cytokeratin 8 (CK8Ser-431) was phosphorylated in the vast majority of CK-expressing VSMC of coronary artery lesions. Only a subset of these cells demonstrated phosphorylation of CK18Ser-33 or, to an even lesser extent, CK8Ser-73. DNA fragmentation occurred predominantly in samples containing cells with phosphorylated CK8Ser-431 domains. In contrast, occluded peripheral lesions exhibited little or no phosphorylation. Neonatal VSMC in umbilical cord vessels contain abundant phosphorylated CK domains, again predominantly CK8Ser-431, but also CK18Ser-33. Again, only single cells were found to be proliferating or to contain DNA fragmentation. Thus, abundant CK phosphorylation in VSMC of atherosclerotic lesions suggests a specific functional response to cell stress and a possible relation to apoptosis.