The biosynthesis of three Met-enkephalin-containing peptides was studied in the hypothalamo-hypophysial system of normal and lactating female rats by the techniques of in vivo radiolabeling and sequential high-performance liquid chromatographic purification of radiolabeled peptides. Met-enkephalin biosynthesis was stimulated in the lactating animals; however, the products derived from the post-translational processing of the enkephalin precursor appeared to remain constant between the two states, as judged by the ratio of radioactivity present in Met5-enkephalin, Met5Arg6Gly7Leu8-enkephalin and Met5Arg6Phe7-enkephalin. Additionally, 8-fold more radioactive enkephalin was harvested from the median eminence than the posterior pituitary. Measurement of preproenkephalin mRNA in the two states also revealed an increase during lactation. All of these data suggest a physiologically important role for the enkephalins in the regulation of anterior pituitary function.