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Preincubation of synaptosomes in standard physiological medium stimulates 2-fold Ca2+ uptake as compared to non-preincubated synaptosomes. When the sodium concentration in the preincubation medium has been halved, Ca2+ uptake was reduced by approximately 50 percent. The addition of ouabain to the preincubation medium decreases depolarization-stimulated Ca2+ uptake by about 40 percent. A steady-state level of Ca2+ uptake is achieved by synaptosomes preincubated for 0, 5 or 10 min. These findings suggest that Ca2+ uptake might depend on the Na-gradient formed during the preincubation of synaptosomes under control conditions.