The factors regulating Ca 2+ transport by isolated sarcoplasmic reticulum (SR) vesicles have been studied using the fluorescent indicator Fluo-3 to monitor extravesicular free [Ca 2+]. ATP, in the presence of 5 mM oxalate, which clamps intravesicular [Ca 2+] at approximately 10 μM, induced a rapid decline in Fluo-3 fluorescence to reach a limiting steady state level. This corresponds to a residual medium [Ca 2+] of 100 to 200 nM, and has been defined as [Ca 2+] lim, whilst thermodynamic considerations predict a level of less than 1 nM. This value is similar to that measured in intact muscle with Ca 2+ fluophores, where it is presumed that sarcoplasmic free [Ca 2+] is a balance between pump and leaks. Fluorescence of Fluo-3 at [Ca 2+] lim was decreased 70% to 80% by histidine, imidazole and cysteine. The K 0.5 value for histidine was 3 mM, suggesting that residual [Ca 2+] lim fluorescence is due to Zn 2+. The level of Zn 2+ in preparations of SR vesicles, measured by atomic absorption, was 0.47±0.04 nmol/mg, corresponding to 0.1 mol per mol Ca-ATPase. This is in agreement with findings of Papp et al. (Arch. Biochem. Biophys., 243 (1985) 254–263). Histidine, 20 mM, included in the buffer, gave a corrected value for [Ca 2+] lim of 49±1.8 nM, which is still higher than predicted on thermodynamic grounds. A possible ‘pump/leak’ mechanism was tested by the effects of varying active Ca 2+ transport 1 to 2 orders with temperature and pH. [Ca 2+] lim remained relatively constant under these conditions. Alternate substrates acetyl phosphate and p-NPP gave similar [Ca 2+] lim levels even though the latter substrate supported transport 500-fold slower than with ATP. In fact, [Ca 2+] lim was lower with 10 mM p-NPP than with 5 mM ATP. The magnitude of passive efflux from Ca-oxalate loaded SR during the steady state of [Ca 2+] lim was estimated by the unidirectional flux of 45Ca 2+, and directly, following depletion of ATP, by measuring release of 40Ca 2+, and was 0.02% of V max. Constant infusion of CaCl 2 at [Ca 2+] lim resulted in a new steady state, in which active transport into SR vesicles balances the infusion rate. Varying infusion rates allows determination of [Ca 2+]-dependence of transport in the absence of chelating agents. Parameters of non-linear regression were V max=853 nmol/min per mg, K 0.5(Ca)=279 nM, and n H(Ca)=1.89. Since conditions employed in this study are similar to those in the sarcoplasm of relaxed muscle, it is suggested that histidine, added to media in studies of intracellular Ca 2+ transients, and in the relaxed state, will minimise contribution of Zn 2+ to fluophore fluorescence, since it occurs at levels predicted in this study to cause significant overestimation of cytoplasmic free [Ca 2+] in the relaxed state. Similar precautions may apply to non-muscle cells as well. This study also suggests that [Ca 2+] lim in the resting state is a characteristic feature of Ca 2+ pump function, rather than a balance between active transport and passive leakage pathways.