Summary The present study aims to validate the technique of in vitro ultrasonography (US) by comparative analysis of premortem intima–media thickness (IMT), postmortem IMT and average wall thickness. In vivo common carotid artery (CCA) IMT was measured bilaterally in 25 patients at 30mm proximal from the flow divider. After autopsy in vitro US was performed and postmortem IMT was measured at the same level. Snap frozen arterial specimens were processed for average wall thickness determination and for histology. High degree of correlation was found between in vivo IMT, in vitro IMT and average wall thickness. Our results demonstrate: (1) in vitro US is a reliable and reproducible tool for the examination of autopsied arterial specimens to obtain valuable information about vascular wall properties and to identify the optimal vascular segment for tissue sampling; (2) snap freezing and cryosectioning of in toto excised arterial specimens is recommended for comparative histological–US studies.