Prolactin secreted by rat anterior pituitary explants into organ culture medium was purified by salt fractionation and gel filtration. A yield of 22 mg/g was obtained, which clearly represented de novo synthesis and secretion of the hormone. Comparative characterization studies were performed on the secreted prolactin and pituitary extracted rat prolactin obtained from the National Institute of Arthritis, Metabolism and Digestive Disease. The biological and immunological activity estimates of both forms were comparable, although the specific activities of the secreted prolactin were somewhat lower than those of the pituitary prolactin. The secreted and extracted forms of prolactin appeared to be identical in primary structure as evidenced by similar amino acid compositions and identical NH2-terminal sequences. Circular dichroism spectra suggested that there may be differences in tertiary structure, since the positive tryptophan band at 292 nm, which eas observed with extracted hormone, was absent in the secreted prolactin.