The relationship between alpha 1-adrenergic receptor occupancy by agonists or antagonists and the regulation of intracellular Ca2+ was examined. Receptor occupancy was measured using the antagonist [3H]prazosin and correlated with agonist-elicited 45Ca2+ fluxes. The agonists epinephrine (E), norepinephrine (NE), and phenylephrine (PE) coordinately activated Ca2+ efflux, reflecting a substantial mobilization of intracellular Ca2+, as well as a smaller 45Ca2+ influx. The agonist concentration dependences for influx and efflux were similar, with the order of potency expected for alpha 1 receptors (E greater than or equal to NE greater than PE). To determine the relationship between receptor occupancy and response, the slowly dissociating antagonist prazosin was used to inactivate specified fractions of the receptor population. A linear relationship was observed between the remaining activatable receptors and residual 45Ca2+ efflux elicited by E or NE, except at saturating agonist concentrations where some curvature was observed. Moreover, the concentration dependence for agonist-elicited 45Ca2+ efflux was shifted toward slightly higher concentrations of E or NE following prazosin inactivation. These results suggest the presence of a modest receptor reserve which is revealed by E or NE, but not by PE. Agonist occupation was measured over the same interval as receptor activation by competition with the initial rate of [3H]prazosin association. All three agonists exhibited the major fraction of receptor occupation over the same concentration ranges required for the functional response. Exposure of receptors to specified agonist concentrations for 30 min had little effect on the number of receptors or their ligand affinities, whereas a 2.5-hr exposure to agonist decreased apparent agonist affinity as well as the number of receptors recognized by [3H]prazosin.