During development of the outflow tract, the walls of the truncus arteriosus change from a diffuse extracellular matrix (ECM) surrounded by an extension of the myocardium to alternating laminae of smooth muscle and elastic connective tissue. The transition rapidly follows septation, when mesenchyme associated with the endothelium differentiates. Using immunocytochemical methods with antibodies to components of the tunica media and the tunica adventitia we have analysed the differentiation of the vessel walls of the outflow tract of the chick. The tunica media marker, elastin, forms laminae in a radial sequence, beginning at the outer margin of the truncus mesenchyme. Conversely, smooth muscle myosin is first expressed in cells associated with the endothelium. Laminin is expressed as a cell surface component throughout the development of the outflow tract. Matrix fibronectin distribution is correlated with the regions that will form the tunica media and apparently forms a radial gradient which is highest near the endothelium. Markers for the tunica adventitia, collagen I and VI, are expressed first at the peripheries of the newly formed tunica media, and collagen VI expression spreads radially through the tunica media. Thus, the vessel wall components appear within the mesenchyme of the truncus arteriosus in opposed radial gradients of differentiation. The tunica media cells acquire secretory and contractile phenotypes independently and may be responding to different stimuli in their expression of these features.