We examined the expression and immunolocalization of water-channel aquaporins in the mammary gland by reverse transcriptase polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemistry. RT-PCR and immunoblotting revealed the expression of aquaporin-1 (AQP1) and AQP3 in the lactating rat mammary gland. AQP3 was detected in the alveolar epithelium and duct system whereas AQP1 was found in the capillaries and venules. AQP3 was present in the basolateral membrane of secretory epithelial cells and intralobular and interlobular duct epithelial cells. The main duct near the orifice in the nipple, which is comprised of a stratified epithelium, bore AQP3 in its basal and intermediate layers. AQP1 was located in both the apical and basolateral membranes of capillary and venule endothelia. AQP3 was not detected in virgin females. AQP3 was found in some differentiating mammary epithelial cells in the pregnant rat. AQP1 was present in capillaries and venules in the differentiating mammary gland of the pregnant rat and in the mammary fat pad of virgin females. We found a similar distribution of AQP1 and AQP3 in the mouse. AQP1 and AQP3 seem to play roles in the synthesis and/or secretion of milk.