Segments of rat carotid artery were maintained in serum-free or serum-supplemented medium for 2 wk, and at intervals of 3, 7, and 14 d the morphology and pattern of matrix synthesis were compared to those in vivo. In serum-free medium and 0.2% serum both the endothelium and the smooth muscle cells (SMC) could be maintained with a minimum of change for 7 d and without substantial change for 14 d. In 2% and 10% serum there was little change for the first 3 d, but subsequently there was a progressive overlapping of the endothelial cells to produce a 3 to 4 layered cell sheet, often separated from the subendothelial matrix; the SMC, however, did not appear to proliferate or migrate and in general retained their typical cellular features for the full time in culture. The synthesis of matrix components was measured by autoradiographic detection of incorporated [H]glucosamine and 35S. At all time periods and serum concentrations the percentage distribution for each label across the arterial wall was found to be similar to that in live animals injected with the same labels. [3H]Glucosamine predominated in the endothelium and the narrow subendothelial layer, which together make up the intima, whereas 35S predominated in the media. In vitro more than 50% of the [3H]glucosamine in the intima and 40% in the adjacent first layer of the media was susceptible to Streptomyces hyaluronidase. As the morphology of both cell types and their synthesis of matrix components could be maintained in organ culture without substantial change we believe that the rat carotid artery may be a suitable model for the investigation of factors affecting arterial structure.