Immunoreactive alpha-melanocyte stimulating hormone (IR-alpha-MSH)-like activity was measured by radioimmunoassay (RIA) in at term pregnancy amniotic fluid prior and after adsorption on a Sep-pak C18 cartridge. alpha-MSH activity was 3-4 times lower after Sep-pak purification but, unlike the levels of IR-alpha-MSH in the fluid analyzed in toto, increased linearly with the volume of fluid analyzed. Furthermore, fractionation by high pressure liquid chromatography (HPLC) revealed that IR-alpha-MSH recovered from the Sep-pak was due to several peptides rather than to a single peptide. The most abundant of them (50% of total activity) behaved like authentic des-acetyl-alpha-MSH. Since des-acetyl-alpha-MSH is also the most abundant alpha-MSH-like peptide in the fetal pituitary gland, the present results suggest that the fetal pituitary is a main source of des-acetyl-alpha-MSH in the amniotic fluid.