The formation of a new blood supply, angiogenesis, is an essential component of carcinogenesis and unrestricted tumor growth. A substance capable of inhibiting angiogenesis would be of considerable therapeutic potential in the treatment of cancer. We previously reported that the 16-kilodalton N-terminal fragment of rat PRL (16K rPRL) was a potent inhibitor of capillary endothelial cell proliferation via a novel receptor. We now report that the nanomolar concentrations of recombinant human 16K PRL inhibit basal and basic fibroblast growth factor- or vascular endothelial growth factor-stimulated growth of bovine brain capillary endothelial cells. 16K human (h) PRL also inhibits stimulation of human umbilical vein endothelial cell proliferation by basic fibroblast growth factor. The organization of endothelial cells into capillary-like structures in type I collagen gels is also prevented by 16K hPRL. Furthermore, in an in vivo assay, the chick embryo chorioallantoic membrane assay, 16K hPRL as well as 16K rPRL were potent inhibitors of capillary formation. 16K hPRL, like 16K rPRL, maintains its biological activity as a partial PRL agonist at PRL receptors on mammary gland epithelial cells. These data demonstrate for the first time that the biological activity of 16K rPRL is not unique and that similar fragments of hPRL are active. The antiangiogenic activity of these molecules is conserved across avian and mammalian species. That 16K hPRL is a potent antiangiogenic factor in in vitro and an in vivo assay raises the exciting potential of this peptide being capable of inhibiting tumor growth.