Collagenase and elastase treatment was used to isolate vascular smooth muscle cells from canine carotid artery. Their structure and function were compared to those in situ. Morphological studies showed that these cells when relaxed in situ were 120–133 µm mean length, connected by numerous typical gap junctions, covered by a basal lamina and like other smooth muscles in structure. After isolation, the median length of single cells was 82 µm. There was structural evidence of some contraction and the basal lamina was absent, but many structures were preserved. Cell clumps of 2–15 cells were often found; cells in such clumps often appeared to be all relaxed or all contracted. Isolated single cells contracted to KCl elevation or to norepinephrine up to 49 or 37% of initial length, EC<sub>50</sub> values for contraction by norepinephrine and KCl were 0.4 µM and 40 mM, respectively; norepinephrine maximum contraction was about 35% less than that for KCl. Lightly loaded spirally cut strips from carotid artery were also studied. EC50 values for norepinephrine and KCl were 4 µM and 40 mM and unaffected by removal of the endothelium. Again, maximum contractions to norepinephrine were less than those to KCl. Contraction speeds were similar for isolated cells and intact strips. However, relaxation of maximally contracted isolated cells did not occur within 10 min. We conclude that canine carotid artery smooth muscle cells can be isolated with little structural or functional damage. The large number of gap junctions between cells and the tendency for cells to be isolated in small groups connected by gap junctions suggests that these cells would be useful for study of cell-to-cell coupling between arterial muscle cells.