Tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) are pro-inflammatory cytokines produced during the development of an immune response. Elevated levels of these cytokines are associated with sepsis, reperfusion injury and heart failure. These experiments were carried out to assess the impact of these two cytokines on the regulation of Ca²⁺ and contraction in isolated rat ventricular myocytes. Treated cells were incubated (at 30°C) for either 1, 2 or 3 h in physiological salt solution supplemented with 0.05 ng/ml TNF-α and 2 ng/ml IL-1β and comparison made between control cells which were incubated in cytokine-free salt solution for the same durations. Cytoplasmic Ca²⁺ was measured using fura-2 or fluo-3 and cell shortening recorded optically. Cells were field stimulated at 1 Hz (30°C) and in experiments carried out on cytokine-treated cells, the superfusate included the same concentrations of cytokines used during incubation. Data are presented as mean ± SEM. After 3 h incubation, the magnitude of the Ca²⁺ transient (control 0.24 ± 0.02, treated 0.16 ± 0.01 fluorescence ratio units) and contraction (control 3.8 ± 0.4, treated 1.9 ± 0.4 % of resting cell length) were significantly (P<0.05, t test, n = 21) decreased. There were no significant changes in the time-course of either the global Ca²⁺ transient (fura-2) or the contraction following treatment. Sarcoplasmic reticulum (SR) Ca²⁺ content, as estimated from the amplitude of Ca²⁺ transients evoked by rapid application of 20 mM caffeine, was significantly reduced (to 77 ± 7% of control, P<0.05, t test, n = 21) after 3 h of cytokine treatment, however the half-time of decay of the caffeine-evoked Ca²⁺ transients was unaffected, suggesting little effect of cytokine exposure on Ca²⁺ efflux mechanisms (e.g. Na⁺-Ca²⁺ exchange). Confocal imaging of cells (at 24°C) demonstrated the presence of Ca²⁺ sparks, the frequency of which was 0.63 ± 0.13 s^-1 in control cells. In cytokine-treated cells, spark frequency was significantly elevated to 2.51 ± 0.17 s^-1 (P<0.001, t test, n = 19). Cytokine-induced effects on spark frequency were abolished when cells were skinned in the continued presence of the cytokines. These data suggest that exposure to TNF-α and IL-1β affects gating of the ryanodine receptor, enhances leak of SR Ca²⁺, which would reduce SR Ca²⁺ content and contribute to the negative inotropic effects of TNF-α and IL-1β. Furthermore, it appears that effects on spark frequency are not mediated by the cytokines directly but by downstream mediators that are lost when cells are skinned.