Isolated acini from lactating mouse mammary glands were prepared by collagenase and hyaluronidase digestion of tissue. Mammary tissue or acini incubated in vitro in tissue culture medium or a similar Ringer's solution lost K and gained Na. Intracellular concentrations approached, but did not equal, the concentrations in the external solution. This ion shift was largely prevented by incubating in a solution with ionic composition resembling mouse milk. In paired experiments, incubation with ouabain (1 mM) caused further increases in Na and decrease in K, suggesting that a functional Na+-K+-ATPase was present. Viability of acini was indicated by normal ATP content and morphology. The ion shift in NaCl-based solutions was slower at 0 degrees C than at 37 degrees C, suggesting that the flux is a membrane-regulated process. Under identical procedures, ion shifts did not occur in thymocytes or a cultured mammary cell line but were seen in both lactating and nonlactating mammary tissue. Nonlactating mammary tissue had a high Na and low K concentration in vivo. As predicted by previous models for the mechanisms of milk secretion, intracellular electrolyte content in mammary epithelial cells appears to be responsive to the ion concentration in the extracellular environment.