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Thapsigargin (TG) and cyclopiazonic acid (CPA) have been reported to be potent inhibitors of the sarcoplasmic reticulum (SR) Ca2+uptake in isolated SR vesicles and cells. We have examined the effect of TG and CPA on (1) the Ca2+uptake by the SR in saponinskinned rat ventricular trabeculae, using the amplitude of the caffeine-induced contraction to estimate the Ca2+content loaded into the SR, (2) the spontaneous Ca2+oscillations at pCa 6.6 using force oscillation as the indicator, and (3) the myofilament Ca2+sensitivity in Triton X-100-treated preparations. Inhibition of Ca2+loading by TG and CPA increased with time of exposure to the inhibitor over 18–24 min. TG and CPA produced half inhibition of Ca2+loading at 34.9 and 35.7 μM respectively, when 18–24 min were allowed for diffusion. The spontaneous force oscillations were more sensitive to the inhibitors: 10 μM TG and 30 μM CPA both abolished the oscillations in this time. The myofilament Ca2+sensitivity was not affected by 10 and 300 μM TG or CPA. The results show that the concentrations of TG and CPA necessary to inhibit the SR Ca2+uptake of skinned ventricular trabeculae are much higher than the reported values for single intact myocytes. One reason for this may be slow diffusion of the inhibitors into the multicellular trabecula preparation.