The specific activity of the Na+/K+/Cl cotransporter was assayed by measuring the initial rates of furosemide-inhibitable 86Rb+ influx and efflux. The presence of all three ions in the external medium was essential for cotransport activity. In cultured smooth muscle cells furosemide and bumetanide inhibited influx by 50% at 5 and 0.2 microM, respectively. The dependence of furosemide-inhibitable 86Rb+ influx on external Na+ and K+ was hyperbolic with apparent Km values of 46 and 4 mM, respectively. The dependence on Cl was sigmoidal. Assuming a stoichiometry of 1:1:2 for Na+/K+/Cl-, a Km of 78 mM was obtained for Cl. In quiescent smooth muscle cells cotransport activity was approximately equal to Na+ pump activity with each pathway accounting for 30% of total 86Rb+ influx. Growing muscle cells had approximately 3 times higher cotransport activity than quiescent ones. Na+ pump activity was not significantly different in the growing and quiescent cultures. Angiotensin II (ANG) stimulated cotransport activity as did two calcium-transporting ionophores. A23187 and ionomycin. The removal of external Ca2+ prevented A23187, but not ANG, from stimulating the cotransporter. Calmodulin antagonists selectively inhibited 86Rb+ influx via the cotransporter. Beta-adrenoreceptor stimulation with isoproterenol, like other treatments which increase cAMP, inhibited cotransport activity. Cultured porcine endothelial cells had 3 times higher cotransport activity than growing muscle cells. Calmodulin antagonists inhibited cotransport activity, but agents which increase cAMP or calcium had no effect on cotransport activity in the endothelial cells.