Hepatitis C virus (HCV) infection has certain characteristics that enable it to play an important role in atherosclerosis. Some studies report its association with an increased risk of carotid artery plaque. The aim of this study was to evaluate the presence of HCV genomic sequences and replicative intermediates in plaque tissues. A cohort of consecutive, prospectively recruited patients with HCV infection and chronic ischemic heart disease from the Cardiology, Vascular Surgery and Hepatology Units of a University Hospital in Florence, Italy, were studied. Positive-strand HCV RNA was detected in seven carotid plaque tissues from anti-HCV-positive patients and was not detected in the nine carotid plaque tissues obtained from anti-HCV-negative patients. In three patients, HCV RNA was found in carotid plaque and not in serum. HCV replicative intermediates were detected in three plaque samples. Direct sequencing of HCV RNA from the plaque and serum showed HCV genotypes 2 (five cases) and 1 (two cases). The novel finding of HCV RNA sequences in plaque tissue strongly suggests an active local infection. This in turn makes it conceivable that the virus may exert local action in carotid atherosclerosis.