beta-Agonists cause an inhibition of contractility and a transient stimulation of Na+/K+ pumping in smooth muscle cells of the stomach from the toad Bufo marinus. To determine if the stimulation of Na+/K+ pumping causes changes in intracellular [Na+] ([Na+]i) that might link Na+ pump stimulation to decrease Ca2+ availability for contraction, [Na+]i was measured in these cells with SBFI, a Na(+)-sensitive fluorescent indicator. Basal [Na+]i was 12.8 +/- 4.2 mM (n = 32) and was uniform throughout the cell. In response to isoproterenol, [Na+]i decreased an average of 7.1 +/- 1.1 mM in 3 sec. Since this decrease in [Na+]i could be completely blocked by inhibition of the Na+ pump, or by blockade of the beta-receptor, [Na+]i reduction is the result of occupation of the beta-receptor by isoproterenol and subsequent stimulation of the Na+ pump. 8-Bromoadenosine 3',5'-cyclic monophosphate and forskolin mimicked the effect of isoproterenol, indicating that the sequence of events linking beta-receptor occupation to Na+ pump stimulation most likely includes activation of adenylate cyclase, production of cAMP, and stimulation of cAMP-dependent protein kinase. The decrease in [Na+]i is sufficiently large and fast that it is expected to stimulate turnover of the Na+/Ca2+ exchanger in the Ca2+ extrusion mode, thereby accounting for the observed linkage between stimulation of the Na+/K+ pump and inhibition of contractility in response to beta-adrenergic agonists.