Milk-lipid droplets are secreted from mammary epithelial cells with an outer envelope of membrane, called the milk lipid globule membrane, which consists of a phospholipid bilayer and protein coat. Constituents of this protein coat, including butyrophilin 1a1 (Btn1a1) and xanthine dehydrogenase/ oxidase (Xdh/Xo), are presumed to function in milk-lipid secretion. We postulate that Btn1a1 is targeted to the apical membrane and binds to Xdh/Xo, which is present on the surface of intracellular lipid droplets. To determine the distribution of Btn1a1 and Xdh/Xo in lactating mammary tissue, we introduced recombinant adenoviral vectors encoding Btn1a1 or Xdh/Xo fused to variants of green fluorescent protein (GFP) into mouse mammary gland. The distribution of the respective expressed fusion proteins was determined by confocal microscopy. Results showed that Btn1a1 and Xdh/Xo were targeted to the apical plasma membrane and the surface of intracellular lipid droplets, respectively. Study of the distribution of either fusion proteins in the secreted droplets showed that the membrane forms crescent-like and blebbing structures, thus confirming earlier electron microscopic data, which suggested that the milk-lipid droplet membrane undergoes structural rearrangement after secretion.