Mammary development and the rate of milk secretion are regulated by frequency and completeness of milk removal. This regulation occurs through chemical feedback inhibition by a milk constituent. Novel, immunologically related milk proteins able to perform this function have been isolated from caprine, bovine and human milk, based on their ability to inhibit milk constituent synthesis in mammary tissue and cell cultures, and to decrease temporarily milk secretion when added to milk stored in the mammary gland. Inhibition is concentration-dependent, suggesting that milk accumulation and removal is accompanied by cyclical changes in inhibitor accretion and depletion in milk. Feedback inhibition is an autocrine mechanism: the caprine inhibitor, termed FIL (feedback inhibitor of lactation) is synthesized by mammary epithelial cells in primary culture. Inhibition is by reversible blockade of the secretory pathway, an effect which, by down-regulating cell-surface hormone receptors, has longer-term consequences on epithelial cell differentiation. Treatment of goat mammary epithelial cell cultures with caprine FIL initially decreased milk protein secretion and subsequently reduced milk protein messenger RNA abundance. Thus the actions of a single milk constituent can bring about both the effect of milking frequency on milk secretion rate and a sequential modulation of cellular differentiation which acts to sustain the secretory response. Long-term regulation, through changes in galactopoietic hormone receptors, also provides an efficient mechanism for integrating acute intramammary regulation of lactation with strategic endocrine control of mammary tissue development.