Leu-Enkephalin (LE) is an endogenous opioid peptide that can arise from two distinct precursors: proenkephalin and prodynorphin. Experiments were designed to differentiate LE derived from proenkephalin versus that derived from prodynorphin. The most dense collections of dynorphin-positive fibers and terminals are in the substantia nigra and posterior pituitary, areas rich in dynorphin-related peptides. The concentration of LE in these regions is significantly higher than that of ME-Arg-Gly-Leu; the ratio of LE to ME-Arg-Gly-Leu is therefore greater than that found in the proenkephalin precursor, which is unity. Globus pallidus deafferentation resulted in a significant decrease of dynorphin B and LE, but not ME-Arg-Gly-Leu, in the substantia nigra. Mild intermittent foot shock (0.2 mA, 20 min) causes a significant increase of dynorphin B and LE in the substantia nigra, but has no effect on ME-Arg-Gly-Leu concentrations. Thus, in the substantia nigra LE may be derived primarily from prodynorphin. Likewise, in the posterior pituitary, osmotic stimulus (e.g., 2% NaCl as drinking fluid) causes marked depletion in dynorphin and LE but has no effect on ME levels suggesting that in the posterior pituitary LE is derived primarily from dynorphin.