Tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) serum levels are increased during the development of sepsis, reperfusion injury and heart failure and these agents either together or alone induce negative inotropic effects on the heart. The experiments described here were to assess the ability of isolated rat ventricular myocytes treated with these two cytokines, to regulate cytosolic Ca²⁺ and contraction during two inotropic interventions: (i) an increase in stimulation rate and (ii) elevation of extracellular Ca²⁺. Treated cells were incubated (at 30 ^οC) for 3 hr 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 duration. Cells were loaded with fura-2 to record cytoplasmic Ca²⁺ and cell shortening was recorded optically. Cells were field stimulated (1 or 3 Hz) at 30 ^οC and for treated cells, the superfusate included the same concentrations of cytokines used during incubation. Sarcoplasmic reticulum (SR) Ca²⁺ content was estimated from the peak of the Ca²⁺ transient evoked by rapid application of 20 mM caffeine. Data are presented as mean ± SEM. In control cells (1 Hz stimulation, extracellular Ca²⁺ 1 mM) the Ca²⁺ transient and contraction were 0.24 ± 0.02 fluorescence ratio (Fr) units and 3.8 ± 0.4% of resting cell length, respectively (n = 24). In treated cells these parameters were reduced significantly (P<0.05, t test) to 0.16 ± 0.01 Fr units and 1.9 ± 0.4% of resting cell length, respectively, n = 24). Increasing stimulation rate to 3 Hz significantly increased (P<0.001, paired t test,) the magnitude of the Ca²⁺ transient, contraction and sarcoplasmic reticulum (SR) Ca²⁺ content in both control (n = 21-27) and treated cells (n = 21-29); however these three variables were all still significantly lower (P<0.007, t test) in treated than control cells. At 3 Hz, the time to peak and time for half relaxation of the Ca²⁺ transient and contraction were significantly accelerated compared to 1 Hz in both cell types (P<0.05, paired t test) but there were no additional cytokine-dependent changes in timecourse. Elevating extracellular Ca²⁺ from 1 to 3 and 5 mM (at 1 Hz stimulation) increased Ca²⁺ transient and contraction amplitude in both control and treated cells (P<0.05, ANOVA, n = 16 & 21, 14 & 21 and 9 & 17 at 1, 3 and 5 Ca²⁺, respectively); however, at both 3 and 5 mM Ca²⁺ we observed no significant cytokine-induced depression of contraction or the Ca²⁺ transient. These data suggest that treated cells have a positive inotropic response to increasing stimulation rate but that contraction amplitude remained below that of control cells. However, elevation of extracellular Ca²⁺ abolished the inhibitory effects of TNF-α and IL-1β on the Ca²⁺ transient and contraction, illustrating a pivotal role for Ca²⁺ regulation in the negative inotropic effect of these cytokines.