Introduction The movements of intra-cellular calcium (Ca) must be fine-tuned in the heart, in order for it to function normally. Disregulation of Ca handling is well known to underlie arrythmogenic activity and contractile dysfunction. We are investigating changes in cellular calcium homeostasis which occur upon the acute application of cardiotoxic drugs. The anti-psychotic clozapine is being used as a model of such a drug (previously associated with cases of myocarditis, arrhythmia and sudden cardiac death). Such in vitro analysis may highlight important drug-screening targets for the future. Methods Rat ventricular myocytes were isolated and stimulated under voltage clamp, inducing systolic Ca transients. Cytosolic calcium was measured using the Ca-sensitive indicator fluo3-AM or FURA-2. Sarcoplasmic reticulum (SR) Ca content was quantified by application of 10 mM caffeine. Rates of decay of calcium transients were used to assess calcium extrusion from the cytosol, by the sarco-endoplasmic reticulum calcium ATP-ase (SERCA) and by sarcolemmal pathways. In order to detect any direct effect of clozapine on ICaL, a step voltage protocol was used to assess changes in the current-voltage (IV) relationship. Phospholamban knockout mice (PLN-KO) were used to ascertain whether effects of the drug on SERCA were phospholamban-dependent. Results 10 μM clozapine significantly reduces peak ICaL (mean reduction 49.7% S.E.M. 1.8%, p <0.001, one way RM ANOVA, n=20). Ca transient amplitude is also significantly reduced (mean reduction 47.7% S.E.M. 2.5%, p <0.001, one way RM ANOVA, n=19). 10 μM clozapine significantly reduces SR calcium content (mean reduction 14.7% S.E.M. 4.7%, p <0.001, student’s t-test, n=11) and SERCA activity (mean reduction 30.5% S.E.M. 2.6%, p <0.001, one way RM ANOVA, n=18). IV results support the findings that 10 μM clozapine significantly and directly affects ICaL. Results from PLN-KO mice show that the effect of clozapine on SERCA is not phospholamban-dependent. Discussion Overall, clozapine’s most noticeable effect is a reduction of peak ICaL, which we speculate could increase the risk of arrhythmogenic activity in the whole heart by introducing refractory heterogeneity. Current work is focusing on the effects of clozapine on calcium spark activity in the quiescent cardiac myocyte.