Murine milk contains 18 mg/ml serum albumin, a concentration equal to that in the serum of the lactating mouse. We examined cellular transport using in vivo methods in the mouse. At steady state the specific activity of 125I-albumin injected into the blood stream was equal in plasma and whey, confirming that milk albumin is extra mammary in origin. Fluorescent albumin crossed the gland from basolateral surface to lumen via cytoplasmic vesicles, but was not transported in the apical to basal direction. Albumin was segregated from transferrin at the basal surface of the epithelial cells and did not colocalize with either caveolin 1 or 2. Vesicular transport was not disrupted by filipin providing additional evidence that, unlike the vascular endothelium, caveoli are not involved. Cytoplasmic albumin was localized to vesicles containing IgA and transport was disrupted by agents that interfere with clathrin mediated endocytosis. Together, these findings provide evidence that albumin is transported across the mammary epithelium by the same pathway as immunoglobulin. The possibility that the massive transfer of albumin into mouse milk is mediated by fluid phase transport is considered.