Utilizing a novel protocol to study transport of substances into mouse milk in situ, we have shown that many "fluid-phase" markers are taken up by mammary epithelial cells and deposited in milk. Since the tight junctions are closed and impermeable even to small molecules, extra-alveolar substances (those not synthesized by the alveolar cells) must be transported into the milk by the epithelial cells themselves. The markers we have used include dextran, lucifer yellow dye, horseradish peroxidase, and albumin. Using these markers and immunostaining for endogenous proteins, we have visualized transcytotic vesicles involved in transporting these markers to milk.