To understand calcium regulation in smooth muscle, we studied both potassium- and norepinephrine-mediated alterations in the movement of calcium in the smooth muscle of rat vas deferens. We employed 45Ca to measure agonist-mediated calcium influx and efflux, as well as tissue calcium content. In addition we labeled tissues with [3H]myoinositol to measure the effect of norepinephrine on inositol phosphate generation. Stimulation of the vas deferens with 50 mM potassium caused a rapid influx of 45Ca (6-fold). Norepinephrine stimulation, even at a concentration maximal for contraction of the tissue (1 mM), did not result in any alteration in 45Ca influx by itself but inhibited potassium-stimulated 45Ca influx (IC50 = 3 microM). This alpha receptor-mediated effect of norepinephrine was not diminished by either pretreatment with reserpine or adrenergic denervation. Studies of the efflux of 45Ca from vas deferens revealed that efflux was not affected by potassium but was significantly stimulated by norepinephrine. Alpha receptor stimulation of vas deferens smooth muscle caused a marked elevation in the appearance of inositol phosphates, particularly inositol trisphosphate, that was not dependent on extracellular calcium. We conclude that norepinephrine does not stimulate calcium influx in vas deferens smooth muscle but leads to the release of calcium from intracellular stores via formation of inositol trisphosphate and that the resulting increase in intracellular calcium may lead to inactivation of the potential-dependent calcium channel.