This study was designed to examine how smooth muscle cell (SMC) isolation affects the distribution of some adherens junctions (AJ) complex associated proteins. Immunofluorescence procedures for identifying protein distribution were used on gastrointestinal and tracheal smooth muscle (SM) tissues and freshly isolated SMCs from dogs and rabbits. As confirmed by force measurements, neither relaxation, Ca⁺⁺ depletion, nor cholinergic activation of SM tissues causes significant redistribution of the AJ associated proteins vinculin, talin or fibronectin away from the plasma membrane. Unlike SMCs in tissue, freshly isolated SMCs show a variable peripheral/cytoplasmic vinculin and talin distribution that is not altered by activation. Enzymatic treatment of SM tissues (as done for the first step of SMC cell isolation) results in loss of fibronectin immunoreactivity in SMCs still in the tissue, but fails to cause redistribution of vinculin, talin or caveolin away from the periphery. The loss of fibronectin immunofluorescence with enzymatic digestion correlates significantly with loss of tissue force production. These results confirm that the AJ associated proteins vinculin and talin do not redistribute throughout SMCs in tissues when relaxed, generating force or following enzymatic digestion. In addition, in freshly isolated SMCs, the distribution of these proteins is significantly altered in approximately 50% of the SMCs. The cause of this redistribution is currently unknown, as is the impact on intracellular signaling and mechanics of these cells. Use of these systems (SMCs in tissues vs. freshly isolated SMCs) provides an ideal situation for studying the role of the AJ in SMC signaling and mechanics.