Butyrophilin 1a1 (Btn1a1), which is a member of the Ig superfamily, is highly expressed in the lactating mammary gland and is secreted into milk in association with lipid droplets. To determine the potential function of Btn1a1 in milk secretion, we ablated Btn1a1 in mice and analyzed the lactation phenotype of homozygous (Btn1a1(-/-)) animals. Two mutant mouse lines were generated in which expression of Btn1a1 was either disrupted or eliminated, respectively. The regulated secretion of milk-lipid droplets was severely compromised in both mutant mouse lines in comparison to wild-type animals. Large pools of triacylglycerol accumulated in the cytoplasm of secretory cells, and lipid droplets escaped from the apical surface with disrupted outer membranes. Luminal spaces became engorged with unstable lipid droplets, which coalesced to form large aggregates. The amount of lipid (wt/vol) was elevated, on average by 50%, during the first 10 days of lactation, and the diameter of the droplets was up to seven times larger than the normal diameter. In contrast, there was no significant difference between wild-type and null animals in the relative amounts of skim-milk proteins secreted from Golgi-derived secretory vesicles. Approximately half the pups suckling Btn1a1(-/-) animals died within the first 20 days, and weaning weights for the surviving pups were 60-80% of those suckling wild-type mice. Thus, expression of Btn1a1 is essential for the regulated secretion of milk-lipid droplets. We speculate that Btn1a1 functions either as a structural protein or as a signaling receptor by binding to xanthine dehydrogenase/oxidase.