When isolated rat myometrium vesicles highly enriched in plasma membranes were preincubated with 100 mM NaCl and then diluted 21-fold in Na-free media, an ATP-independent Ca uptake value of 4.10 +/- 0.23 mumol/g protein occurred, compared to a value of 2.87 +/- 0.16 for a similar uptake by vesicles preincubated in Na-free media. Brief (less than 10 s) exposure of the membrane vesicles to 5 mM ethyleneglycol-bis(beta-aminoethyl)-N,N'-tetraacetic acid (EGTA) after the Ca uptake showed that the NaCl preincubated vesicles retained more Ca than the sucrose or KCl preincubated vesicles. A NaCl concentration in the membrane fractions identical to its concentration in the Ca uptake medium did not enhance the Ca uptake by the vesicles did not show an increased Ca uptake. NaCl added to plasma membrane vesicles actively loaded with Ca caused retention of less Ca than the control. NaCl added to actively loaded vesicles along with EGTA also enhanced calcium efflux compared to EGTA alone. Sucrose, K+, Rb+, or Cs+ could not replace Na+ for the Na+-dependent Ca uptake or release, while Li+ was a poor substitute in both the instances. Na+-dependent Ca-uptake distribution in the various fractions correlated very well with their 5'-nucleotidase activity but not with their NADPH- or succinate-dependent cytochrome c reductase activities. The results have been discussed using a Na--Ca exchange model as well as by a model in which Na+ competes for calcium binding to the membranes.