Fibrinogen is a complex plasma protein composed of two each of three different polypeptide chains. We have targeted expression of r-human fibrinogen to the mammary gland of transgenic mice. Three expression cassettes, each containing the genomic sequence for one of the three human fibrinogen chains controlled by sheep whey protein beta-lactoglobulin promoter sequences, were coinjected into fertile mouse eggs. Southern blot analysis demonstrated that more than 80% of the transgenic founders contained all three fibrinogen genes. Reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis of milk from the highest producing founder animal demonstrated the presence of human fibrinogen subunits at concentrations of 2000 micrograms/ml. In several animals with a balanced ratio of the individual fibrinogen subunits, up to 100% of the protein was incorporated into fully assembled fibrinogen hexamers. Incubation of the transgenic milk with thrombin and factor XIII resulted in a cross-linked fibrin clot, indicating that a major portion of the secreted fibrinogen was functional. These studies represent the first report of high-level biosynthesis and secretion of a functional, complex, hexameric protein in the milk of a transgenic animal.