Intracellular calcium (Ca) dynamics are finely tuned in the healthy heart. This homeostasis may be interrupted during a response to a cardiotoxic substance, leading to contractile dysfunction and arrhythmogenic activity. Here we aim to investigate the changes in cellular Ca homeostasis occurring upon acute application of the cardiotoxic drug clozapine (atypical anti-psychotic). Clozapine use has previously been associated with cases of myocarditis, arrhythmia and sudden cardiac death. Such in vitro analysis may highlight important new drug-screening targets for the future. Ventricular myocytes were isolated from male rats (200-250g). Myocytes were perforated-patched and stimulated under voltage clamp control (1mM external Ca), inducing systolic Ca transients. Ca handling parameters were measured both in control conditions and upon acute application of 10μM clozapine. Cytosolic Ca was quantified by Ca-sensitive indicators fluo-3 or fura-2. 10mM caffeine was applied to discharge the sarcoplasmic reticulum (SR) and quantify SR Ca content. Changes in cytosolic Ca extusion (SERCA – sarco-endoplasmic reticulum ATP-ase, NCX – sodium-calcium exchanger) were calculated by comparing the decay rate of Ca transients. The protocol was repeated in cells from a phospholamban knockout mouse model (PLB-KO) in order to assess whether any effects were PLB-dependent. In a protocol to induce spontaneous SR Ca release and waves, external Ca was elevated to 5mM; immediately after a wave 10mM caffeine was applied to discharge the SR. Threshold for spontaneous Ca release was calculated by combining SR content value with the amount of Ca leaving the cell on the wave. Statistical tests used were one way RM ANOVA and student’s t-test. Data presented as mean ±SEM. Clozapine reduced mean Ca transient amplitude by 47.7±2.5% (p<0.001 n=19) and peak L-type Ca current (ICaL) by 49.7±1.8% (p<0.001 n=20). SR content was decreased by 15.5±4.2% (p=0.004 n=9), SERCA activity (rate constant) by 33.9±2.6% (p<0.001 n=16) and NCX rate constant by 16.6±3.8% (p=0.005 n=6). The effect of clozapine on SERCA persisted in PLB-KO indicating that the effect on SERCA is not PLB-dependent. Results suggest that clozapine does not significantly alter the threshold for spontaneous SR Ca release and waves. Acute application of clozapine to isolated myocytes causes several changes to Ca dynamics, most noticeably a reduction in peak ICaL. It remains unclear if or how the observed effects on Ca handling contribute to the clinical cardiotoxicity of clozapine. One possibility is that the effects on ICaL predispose to refractory heterogeneity across the heart. We also speculate that in the clinical setting there may be accumulation of clozapine in cardiac myocytes over time, supported by our results that the effect of clozapine on systolic Ca transient amplitude is not fully reversible upon wash-out of the drug.