Measurements on the directional organization of collagen are necessary for relating the structure and mechanical function of blood vessels. The birefringent optical property of collagen has enabled us to assess the collagen architecture for brain arteries, which are prone to spasm and aneurysm formation. Using the universal stage and polarizing microscope, we measured the three-dimensional organization of the collagen of the main layers of the artery wall, and examined the effect of distending pressure on that organization. Adult arteries obtained from autopsy were fixed at one of three distending pressures, 30, 120 and 200 mm Hg; they were embedded in paraffin and sectioned parallel to the vessel axis at 4 microns thickness. Sections were stained with picrosirius red, a birefringent enhancement stain specific for collagen. Orientation data were obtained from tangential sections from thirteen arteries. We chose to use tangential sections that graze the curving surface of individual layers, to permit measurements that are equally sensitive to fibres in the mechanically meaningful range of directions including longitudinal, helical and circumferential. Each measurement was from a single fibre or group of fibres at a specific location; the mean direction and its variation of alignment within each artery layer were calculated. In some arteries, the adventitia and subendothelium measurements were separated into sublayers, distinguishable by the birefringent optical appearance. Main findings included a substantial component of longitudinal fibres in the adventitia and subendothelium, highly varied in coherence and mean direction, and a thin collagen layer of the adventitia, radially outside the medial muscle cells, that was highly organized circumferentially (circular standard deviation of 9 degrees). At higher pressures, the collagen fabric of all the layers was increasingly coherent and more circumferential in direction.