Sarcoplasmic reticulum (SR) Ca content is important in determining the size of the systolic Ca transient. The increase of systolic Ca transient amplitude produced by β-adrenergic stimulation results, at least in part, from an increase of SR Ca. One well-established mechanism for the increase of SR Ca is due to phosphorylation of phospholamban thus increasing SR Ca uptake by the SR Ca-ATPase (SERCA) (Tada et al, 1974). We have investigated whether other mechanisms can contribute to the increase of SR Ca. Ventricular myocytes were isolated from wild-type (WT) (C57Bl/6J) and phospholamban knockout (PLN^-/-) mice killed by cervical dislocation. Whole cell patch clamp studies were performed at 37°C using a Cs⁺ based pipette solution containing the Ca indicator Fluo-5F. Myocytes were voltage clamped and stimulated with depolarising steps to 10 mV from a holding potential of -60 mV. SR Ca content was measured by integrating the sodium-calcium exchange (NCX) current produced by rapid application of 10 mM caffeine. Data are presented as mean ± S.E.M with the Students t-test used for statistical analyses. β-adrenergic stimulation (100 nM isoprenaline) increased the systolic Ca transient amplitude in WT and PLN^-/- myocytes by 124±25% and 85±30% respectively. This was accompanied by an increase of the amplitude of the L-type Ca current from 11.8±0.8 to 14.9±1.0 pA/pF (n=12 myocytes) in WT and 9.1±0.9 to 12.9±1.2 pA/pF (n=14 myocytes) in PLN^-/-. SR Ca content increased in WT from 69±2 to 86±5 µmol.l^-1 (p<0.01, n=12 myocytes). In contrast, in PLN^-/- myocytes, β-adrenergic stimulation decreased SR Ca content from 174±17 to 127 ±12 µmol.l^-1 (p<0.01, n=14 myocytes). These results show that phosphorylation of phospholamban is required for the increase in SR Ca content in response to β-adrenergic stimulation. Previous work (Trafford et al, 2001) has shown that increasing the L-type Ca current (by increasing external Ca concentration) decreases SR Ca content. This result was attributed to the fact that although an increase of L-type Ca current increases Ca entry into the cell, it also triggers more Ca release from the SR and thence more Ca efflux from the cell tending to deplete the SR. A similar mechanism can account for the observed decrease of SR Ca content during β-adrenergic stimulation in the absence of phospholamban. We conclude that β-adrenergic stimulation results in a net increase in SR content due to the effects of phospholamban phosphorylation.