Time-dependent variations in the vasodilator effects of β-adrenergic agonists terbutaline (Ter) and dobutamine (Dob) were studied in isolated rings of rat thoracic aorta in both endothelium-intact and endothelium- denuded preparations. Rats were housed in light from 08:00 to 20:00 and in darkness from 20:00 to 08:00 and sacrificed at six different times of the day. In endothelium-intact and endothelium-denuded aortic rings precontracted submaximally with phenylephrine (Phe), addition of Ter and Dob produced concentration-dependent relaxations. Removal of endothelium reduced the relaxant responses and area under curve (AUC) values and augmented the EC50 values to Ter and Dob at most, but not all, time points. Two-way analysis of variance (ANOVA) revealed that AUCs, maximum responses, and EC50 values significantly depended on both treatment (endothelium intact/endothelium denuded) and time of sacrifice. Results of the present study clearly show that in vitro sensitivity of rat thoracic aorta to β-adrenergic agonists displays temporal variations depending on the time of animal sacrifice, and the presence of endothelium modifies the rhythmicity in β-adrenergic activity. These variations may be due to the circadian rhythmicity in the adenylyl cyclase-cAMP-phosphodiesterase system that mediates the responses to β-adrenergic agonists.