In cardiac myocytes calcium release occurs by the process of calcium induced calcium release (CICR). The action potential activates the L-type Ca current and the resulting Ca influx triggers the opening of the sarcoplasmic reticulum (SR) Ca release channel (ryanodine receptor, RyR). However, if the amount of Ca stored in the SR reaches a critical or threshold concentration, waves of CICR can occur during diastole independent of the action potential (also known as diastolic Ca release). Beta adrenergic stimulation produces Ca waves. It has been suggested that this occurs by phosphorylation of the RyR that increases its activity and lowers the SR threshold for diastolic Ca release.(Marx et al., 2000). An alternative explanation, however, of the ability of beta adrenergic stimulation to produce Ca waves is via an increase of SR Ca content secondary to phosphorylation of phospholamban and thence stimulation of the SR Ca-ATPase (SERCA). The main objective of this study was to directly measure the effects of beta adrenergic stimulation on the threshold for diastolic Ca release. Experiments were performed in rat ventricular myocytes patch clamped using the perforated patch technique. Cells were held at -40 mV and stimulated at 0.5 Hz using voltage steps to 0 mV at room temperature. Diastolic Ca waves were induced by applying high external Ca (4mM) and long depolarizing steps (500 msec). 1 µM isoprenaline was applied to test the effects of beta adrenergic stimulation. When stimulation was stopped a few Ca waves were observed immediately after the last Ca transient. Caffeine (5 mM) and Butanedione Monoxime (10 mM) were applied immediately after the last Ca wave in both conditions to determine the threshold SR Ca content for diastolic Ca waves. The integral of the caffeine induced current was used to quantify SR Ca content. The threshold for spontaneous Ca release was significantly higher in the presence of isoprenaline ( 94±15 µmol/l in 4 Ca and 123±14 µmol/l in 4 Ca + isoprenaline; n=8, P=0.001, paired t test). On the basis of these data we conclude that beta adrenergic stimulation increases the SR threshold for spontaneous Ca release. This result is not what would be expected from phosphorylation of the RyR. It may be due to stimulation of SERCA making it more difficult for Ca waves to propagate (O’Neill et al., 2004).