Amino acid transport system A is present in many cells and tissues and is regulated by hormones and other factors, including hypertonic stress. System A in vascular smooth muscle cells is also activated when microtubules are disrupted by drugs such as colchicine. The present study examined the action of colchicine on hypertonic activation of system A in smooth muscle cells. In serum-free medium, activation of system A by modest (340 mOsm) hypertonicity was not affected by colchicine addition. However, at high osmotic stress (460 mOsm) the addition of colchicine partially blocked the activation of system A. Addition of colchicine alone, at normal osmolarity, produced activation of system A. In the presence of serum, colchicine action was markedly different. Colchicine consistently inhibited hypertonic activation of system A at any degree of hypertonic stress but had no effect on system A at normal osmolarity. The action of colchicine as both an activator and inhibitor of system A implies microtubule involvement at more than one step in the intracellular regulation of system A.