Hydrogen peroxide (H₂O₂) can be used to mimic intra and extracellular reactive oxygen species generation in disease states such as sepsis. Goldhaber et al. (1994) studied the effects of H₂O₂ on calcium handling in the ventricular myocyte at room temperature. As the cytoxicity of H₂O₂ is temperature dependent, it is important to understand its effect at physiological temperatures. Male Wistar Rats were humanely killed by cervical dislocation in accordance with The Home Office Schedule 1 regulations. Ventricular myocytes were isolated via enzyme digestion and loaded with Fluo-3AM for cytoplasmic calcium ([Ca²⁺]_i) measurement. Membrane currents were measured via perforated patch under voltage clamp conditions. Cell length was measured via video edge detection. Statistical significance was determined via the students t-test, and a value of less than 0.05 was deemed significant. Data is shown as mean±S.E.M. 200 μM H₂O₂ resulted in a progressive reduction in [Ca²⁺]_i transient amplitude (n=10, Control:272±29, H₂O₂:153±17 nmol/l, p<0.0001) and an increase in diastolic [Ca²⁺]_i (n=10, Control:93±5, H₂O₂:121±7 nmol/l, p<0.0001). Associated with this was a decrease in systolic cell shortening (n=7, Control:2.24±0.43, H₂O₂:0.87±0.14%, p<0.05), a reduced diastolic cell length (n=7, Control:123±8, H₂O₂:120±8 μm, p<0.005) and an increased relaxation time (n=7, Control:526±57, H₂O₂:746±118 ms, p<0.05) consistent with a reduced rate constant of [Ca²⁺]_i decay (n=10, Control:4.63±0.49, H₂O₂:3.79±0.36 s^-1, p<0.005). No change in the peak L-type calcium current was observed. L-type calcium influx was increased (n=10, Control:3.16±0.14, H₂O₂:3.43±0.18 µmol/l), whilst calcium efflux via the sodium calcium exchanger (NCX) was decreased (n=6, Control:1.26±0.13, H₂O₂:1.12±0.10 s^-1, p<0.05). Sarcoplasmic reticulum (SR) content was reduced (n=6, Control:95±11, H₂O₂:69±7 μmol/l, p<0.05), however the diastolic [Ca²⁺]_i rise still occurred with inhibition of SR function. Hyperpolarisation of the membrane voltage to -120 mV reversed the H₂O₂ induced [Ca²⁺]_i rise (n=9, -40 mV:2.57±0.98, -120 mV:-1.35±0.55 nmol/l s^-1, p<0.05). Reversal of [Ca²⁺]_i rise upon hyperpolarisation is consistent with the findings of Song et al (2006) who found H₂O₂ induced an increase in intracellular Na⁺ ([Na⁺]_i). Increased [Na⁺]_i could lead to increased [Ca²⁺]_i via reverse mode NCX activity. These data suggest that H₂O₂ induced SR dysfunction reduces the [Ca²⁺]_i transient amplitude and is independent of L-type calcium current. H₂O₂ induced diastolic [Ca²⁺]_i rise occurs independently of the SR. This diastolic [Ca²⁺]_i rise may be due to increased [Na⁺]_i however increased Ca²⁺ influx combined with a impairment of Ca²⁺ removal mechanisms could be a contributing factor to H₂O₂ induced diastolic dysfunction.