In vitro mammary epithelial cell models typically fail to form a consistently tight barrier that can effectively separate blood from milk. Our hypothesis was that mammary epithelial barrier function would be affected by changes in luminal ion concentration and inflammatory cytokines. BME-UV cells were grown to confluence on permeable supports with a standard basolateral medium and either high electrolyte (H-elec) or low electrolyte (L-elec) apical medium for 14 days. Apical media were changed to/from H-elec at predetermined times prior to assay. Transepithelial electrical resistance (R_te) was highest in monolayers continuously exposed to apical L-elec. A time-dependent decline in R_te began within 24 hours of H-elec exposure. Change from H-elec to L-elec time-dependently increased R_te. Permeation by FITC-conjugated dextran was elevated across monolayers exposed to H-elec, suggesting compromise of a paracellular pathway. Significant alteration in occludin distribution was evident concomitant with the changes in R_te, although total occludin was unchanged. Neither substitution of Na⁺ with N-methyl-D-glucosamine (NMDG⁺) nor pharmacological inhibition of transcellular Na⁺ transport pathways abrogated the effects of apical H-elec on R_te. Tumor necrosis factor alpha, but not interleukin-1 nor interleukin-6, in the apical compartment causes significant decrease in R_te within 8 hours. These results indicate that mammary epithelium is a dynamic barrier whose cell-cell contacts are acutely modulated by cytokines and luminal electrolyte environment. Results not only demonstrate that BME-UV cells are a model system representative of mammary epithelium, but also provide critical information that can be applied to other mammary model systems to improve their physiological relevance.