Na-Ca exchange in the plasma membrane-enriched fraction N1 isolated from rat myometrium has been demonstrated as Ca gain or loss depending on the direction of the [Na+] gradient [Grover et al., Am. J. Physiol. 240 (Cell Physiol. 9): C175-C182, 1981]. In this study, the [Na+]gradient-dependent Ca uptake by this membrane fraction was demonstrated to occur optimally at pH 6.67; and 7.0 +/- 2.2 microMs Ca2+ was required for the half-maximal uptake. Such Ca uptake occurred in vesicles previously loaded with a [Ca] that was the same as or higher than the one present in the Ca-uptake medium, i.e., as an uphill Ca-transport process. The Na-Ca exchange-dependent Ca uptake was abolished by the Ca ionophore A23187 or by the monovalent cation (Na+) ionophore monensin. It was not influenced by the presence of KC1, KC1 plus valinomycin, or the proton uncouplers carbonyl cyanine p-trifluoromethoxyphenyl hydrazone or 5'-chloro-3-t-butyl-2'-chloronitrosalicylanilide in the Ca-uptake medium. The properties of the Na-Ca exchange are compared with those of passive and ATP-dependent Ca uptake by this membrane fraction, and physiological implications of this process are discussed.