Prolactin (PRL) in milk influences maturation of gastrointestinal epithelium and development of both the hypothalamo-pituitary and immune systems of offspring. Here, we demonstrate that most PRL in human milk is part of a novel, high-affinity, multicomponent binding complex found on the milk fat globule membrane and not in whey. To examine properties of the complex, a sensitive ELISA was developed such that human PRL (hPRL) binding to the complex was measured by loss of hPRL detectability; thus, as much as 50 ng of hPRL was undetectable in the presence of 10 μl of human milk. Using the same methodology, no comparable complex formation was observed with human serum or amniotic fluid. hPRL complexation in milk was rapid, time dependent, and cooperative. Antibodies to or competitors of the hPRL receptor (placental lactogen and growth hormone) showed the hPRL receptor was not involved in the complex. However, hPRL complexation was antagonized by cyclosporine A and anti-cyclophilins. The complex was very stable, resisting dissociation in SDS, urea, and dithiothreitol. Western analysis revealed an ∼75-kDa complex that included hPRL, cyclophilins A and B, and a 16-kDa cyclophilin A. Compared with noncomplexed hPRL, complexed hPRL in whole milk showed similar activation of STAT5 but markedly delayed activation of ERK. Alteration of signaling suggests that complex formation may alter hPRL biological activity. This is the first report of a unique, multicomponent, high-capacity milk fat reservoir of hPRL; all other analyses of milk PRL have utilized defatted milk.