We investigated the expression of butyrophilin in eukaryotic cells with a view to determining the number of mRNA species, the incorporation of the peptide chain into microsomes, and the topology of the processed protein in biological membranes. Butyrophilin is synthesized from a single sized mRNA in both bovine and murine lactating mammary tissue and associates with microsomal membranes with a type I topology (N exo ⅐C cyto ) via a single hydrophobic anchor in the middle of the sequence. Several isoelectric variants of the protein were detected in cellular membranes from lactating bovine mammary tissue and in the milk-fat-globule membrane. We found no evidence for soluble forms of butyrophilin in postmicrosomal supernatants. The 66-kDa protein appears to be subjected to limited proteolysis, giving rise to a 62-kDa fragment lacking the C terminus and to other more minor fragments of lower M r in the milk-fat-globule membrane. Antipeptide antibodies to epitopes within the N-and C-terminal domains were used to show that butyrophilin retains a type I topology in plasma membranes when expressed in insect cells from a baculovirus vector, and in secreted milk-fat globules. These data do not agree with previous suggestions that butyrophilin may exist in cytoplasmic soluble forms, or be reorganized in the plane of the lipid bilayer during secretion in lipid droplets from mammary cells. The results are discussed with reference to the role butyrophilin may play as the principal scaffold for the assembly of a complex with xanthine oxidase and other proteins that functions in the budding and release of milk-fat globules from the apical surface during lactation.Several years ago we cloned a cDNA encoding the major milk-fat-globule membrane (MFGM) 1 protein, butyrophilin (BTN) (1) with a view to understanding the function of this mammary-specific protein in milk-fat secretion (for reviews of this process, see Refs. 2-4). From the derived amino acid sequence we predicted that BTN is an integral protein with a single membrane anchor and a glycosylated exoplasmic N terminus (type I orientation) (1). Subsequent comparisons with more recently cloned cDNAs have established that BTN is a member of the immunoglobulin superfamily (5) with two Nterminal immunoglobulin domains, one of the intermediate type (IgI) (6) toward the N terminus and one of the constant C1 type (7) toward the membrane bilayer (8). A large proportion of the C-terminal region comprises the RFP or B30.2 domain, a sequence which is present in a subfamily of zinc-finger proteins (9, 10), and a group of recently identified BTN-like genes (11, 12). Several workers have proposed that BTN may function as an integral receptor for cytoplasmic fat droplets and that budding of the droplets at the cell surface is initiated by interactions between the cytoplasmic tail of BTN and other proteins, notably the redox enzyme xanthine oxidase (1, 13-16). However, the assumption that BTN behaves as an integral protein in mammary epithelial cells has been confounded by several observati...