Epithelial cells dissociated from midpregnant BALB/c mouse mammary glands were cultured for as long as 20 days as confluent monolayers on floating collagen gels. Detached gels bearing monolayers were placed in lucite Ussing chàmbers for measurement of transepithelial potential difference (PD), short-circuit current (I(sc)), resistance (R), and unidirectional fluxes of Na(+) and Cl(-) during short-circuit current conditions (PD = 0). With Hanks' solution bathing both sides of cultures maintained with insulin and cortisol, PD = -12.8 mV (serosal side ground), I(sc) = 24.6 muA/cm(2), and R = 507 omega.cm(2). Net absorption of Na(+) equaled I(sc), and there was no net Cl(-) transport. PD and I(sc) were reduced 50% by mucosal addition of 10 muM amiloride and to zero by metabolic inhibition with nitrogen gas or by serosal addition of 0.1 mM ouabain. In similar cultures supplemented with prolactin, PD and I(sc) increased to -15.8 mV and 48.0 muA/cm(2), respectively, and R decreased to 374 omega.cm(2). Inhibitor effects were similar to those seen in prolactin-free cultures. Prolactin exposure resulted in a 3-fold increase in net absorption of Na(+). Na(+) absorption was not equivalent to I(sc), and there was little Cl(-) absorption; therefore, prolactin induced active transport of other, as yet unidentified, ions. These effects of prolactin require at least 3 days to occur and cannot be attributed to the known contamination with neurohypophysial hormones. The prolactin-induced increase in Na(+) absorption parallels its Na(+)-retaining ability in lower vertebrates and could be part of the mechanism that keeps milk Na(+) concentration low in intact glands.