Immunoreactive rat prolactin was detected in rat milk. Increasing volumes of milk decreased the percentage of 131I-labelled rat prolactin bound to antiserum in a manner parallel to the decreases produced by increasing log doses of standard hormone and blood serum. Rats suckling litters of 4, 8 or 12 pups were milked at various stages of lactation and prolactin concentrations (ng/ml) in milk varied significantly both with stage of lactation and with litter size. Mean prolactin levels were maximal on day 4 to day 15 and they decreased sharply near the end of lactation. At every stage of lactation litter size tended to influence prolactin concentrations with larger litters always being associated with more prolactin per millilitre of milk. Intraperitoneal injection of 1 mg bovine prolactin into lactating rats promptly caused the appearance of high levels of this foreign hormone in rat milk without significantly affecting the concentration of the native hormone.