The location and origin of carotenoids in bovine milk fat globules was investigated using spectral absorption of lipid solutions at 461 nm to quantitate carotene. Release of membrane from globules as a result of churning to butter or by freezing and thawing of the globules yielded membrane preparations which were devoid of carotene. Globule cores from these procedures exhibited carotene concentrations comparable to those in total milk lipids. Fractionation of lactating bovine tissue and analysis of lipid extracts revealed that the intracellular fat droplets have carotene concentrations approximating those of secreted globules. However, intracellular membranes of the tissue, particularly the mitochondria, are much richer in carotenoids than formative or secreted fat globules. The evidence indicates that bovine milk fat globules acquire carotene during their formation in the cell, but that some minor fraction of the total carotene may be extracted from the enveloping secretory membrane. Mean carotene values (microgram/g of lipid) for fractions from three samples of lactating tissue were: whole tissue 47, mitochondria 461, microsomes 69, cytosol 67, fat droplets 8, milk 9. One tissue analysis indicated that Golgi membranes contain somewhat more carotene than do microsomes.