Thapsigargin (TG) and cyclopiazonic acid (CPA) are reported to be specific high-affinity inhibitors of the sarcoplasmic reticulum (SR) Ca2+ pump in isolated membranes and cells, with TG causing complete pump inhibition at nanomolar concentrations. To evaluate the effectiveness of TG and CPA in small multicellular cardiac preparations, we used rapid cooling contractures (RCCs) to assess the SR Ca2+ load. In contrast to observations in single myocytes, TG caused remarkably slow and incomplete SR Ca2+ depletion in multicellular preparations. A 45-minute exposure to 500 μM TG at 30°C and 0.5-Hz stimulation only decreased RCCs by 76±5% (and 100 μM CPA reduced RCCs by 59±10% [mean±SEM]). In contrast, 10 minutes with 20 mM caffeine completely abolished RCCs. This confirms that there was still a caffeine-sensitive pool of Ca2+ in the TG-treated muscle. The time constant of rest decay of RCCs was accelerated by both TG (from 83±18 to 26±6 seconds) and CPA (from 68±11 to 10±5 seconds). This might be expected since Ca2+ leaking from the SR during rest cannot be taken back up as efficiently, favoring Ca2+extrusion by the sarcolemmal Na+-Ca2+ exchanger. TG and CPA decreased twitch force (by 44±7% and 40±11%, respectively) and increased twitch duration, presumably because of the SR effects. We conclude that complete blockade of SR Ca2+ uptake by TG or CPA in multicellular preparations cannot be assumed, even at high [TG] or [CPA], unless evaluated (eg, by RCC).