Radioactive calcium flux in rabbit aorta was studied to evaluate factors regulating the movement of calcium into and out of the cell. Calcium content was estimated both from (1) the amount of 45 Ca present in the tissue and the specific activity of Ca in the bathing medium, and (2) determination of chemical calcium by atomic absorption spectrophotometry. Calcium efflux was followed by washing aortic strips previously loaded with 45 Ca in nonlabeled salt solution. Norepinephrine (10 -6 g/ml) caused a significant increase in the calcium content calculated from radioactive calcium activity, whereas isoproterenol (10 -8 g/ml) caused a significant decrease in calcium content. Measurements of efflux rates, first in calcium-free solution and subsequently in 1.6 m M Ca, demonstrated that norepinephrine delays calcium efflux into calcium-free solution and delays the rapid exchange of calcium that occurs upon exposure of muscle to a calcium-containing salt solution. Isoproterenol produced no significant change in these rates. These observations are compatible with the possibilities that norepinephrine causes contraction by decreasing calcium efflux and that isoproterenol causes relaxation by decreasing calcium influx.