B-mode ultrasound is being used to assess carotid atherosclerosis in epidemiological studies and clinical trials. Recently the interpretation of measurements made from ultrasound images has been questioned. This study examines the anatomical correlates of B-mode ultrasound of carotid arteries in vitro and in situ in cadavers. Twenty-seven segments of human carotid artery were collected at autopsy, pressure perfusion fixed in buffered 2.5% glutaraldehyde and 4% paraformaldehyde and imaged using an ATL UM-8 (10 MHz single crystal mechanical probe). Each artery was then frozen, sectioned and stained with van Gieson or elastin van Gieson. The thickness of the intima, media and adventitia were measured to an accuracy of 0.01 mm from histological sections using a calibrated eye graticule on a light microscope. Shrinkage artifact induced by histological preparation was determined to be 7.8%. Digitised ultrasound images of the artery wall were analysed off-line. The distance from the leading edge of the first interface (LE1) to the leading edge of the second interface (LE2) was measured using a dedicated programme. LE1-LE2 measurements were correlated against histological measurements corrected for shrinkage. Mean values for the far wall were: ultrasound LE1-LE2 (0.97 mm, S.D. 0.26), total wall thickness (1.05 mm, S.D. 0.37), adventitia (0.35 mm, S.D. 0.16), media (0.61 mm, S.D. 0.18), intima (0.09 mm, S.D. 0.13). Ultrasound measurements corresponded best with total wall thickness, rather than elastin or the intima-media complex. Excision of part of the intima plus media or removal of the adventitia resulted in a corresponding decrease in the LE1-LE2 distance of the B-mode image. Furthermore, increased wall thickness due to intimal atherosclerotic thickening correlated well with LE1-LE2 distance of the B-mode images. B-mode images obtained from the carotid arteries in situ in four cadavers also corresponded best with total wall thickness measured from histological sections and not with the thickness of the intima plus media. In conclusion, the LE1-LE2 distance measured on B-mode images of the carotid artery best represents total wall thickness of intima plus media plus adventitia and not intima plus media alone.