Ca2+ uptake by the sarcoplasmic reticulum (SR) and free [Ca2+] were measured simultaneously with indo 1 and a Ca2+-selective minielectrode in suspensions of permeabilized rabbit or rat ventricular myocytes (≈10 mg/mL protein). In the presence of 25 μmol/L ruthenium red and 10 mmol/L oxalate, the K. for Ca2+ uptake by the SR was ≈250 nmol/L in rabbit and rat ventricular myocytes. The maximal Ca2+ uptake rate was 2.4 times higher in rat than in rabbit. Addition of 5 nmol thapsigargin (TG) per milligram cell protein abolished Ca2+ uptake completely in both species. The [TG] necessary for a half-maximal reduction of the uptake rate (K1/2) was 55 pmol/mg cell protein for rabbit and 390 pmol/mg cell protein for rat. Assuming that the number of pump sites is two times the concentration of TG necessary to inhibit half of the Ca2+ pump activity (ie, the TG affinity is very high), the density of pump sites is 7.7 μmol/kg wet wt for rabbit and 54.6 μmol/kg wet wt for rat. Despite a fivefold decrease of the Ca2+ uptake rate by a submaximal [TG], the permeabilized myocytes were still able to lower the free [Ca2+] to <150 nmol/L from a peak value >10 μmol/L. The relative inhibition of Ca2+ uptake by TG did not depend on the free [Ca2+]. Addition of more than 5 nmol TG per milligram cell protein abolished Ca2+ uptake by the SR completely in <15 seconds and reduced the uptake rate by 95% in 5 seconds. We conclude that TG can rapidly and completely inhibit the SR Ca2+ pump and that there may be more pump sites in rat than in rabbit ventricle.