The distribution and projection of immunoreactive gamma-melanocyte-stimulating hormone (gamma-MSHI) in the rat brain was examined by indirect immunofluorescence using an antiserum against synthetic rat gamma-MSH. The present study confirmed the presence of gamma-MSHI neurons in the arcuate nucleus and further demonstrated that the n. commissuralis is a new gamma-MSHI neurons-containing site. We also found a gamma-MSHI fiber network in the hypothalamus, thalamus, amygdala, central gray matter of the midbrain and upper pons, and further demonstrated a much more extensive distribution of these fibers particularly in the medulla oblongata, an area previously thought not to contain gamma-MSHI structures. The present observation on the normal distribution of gamma-MSHI suggested the existence of two different systems: one is the arcuatofugal gamma-MSH system and the other n. commissuralis gamma-MSH system. Using experimental manipulations, we clearly established that gamma-MSHI fibers in the forebrain, diencephalon, midbrain and upper pons originate from gamma-MSHI neurons in the arcuate nucleus and those in the medulla oblongata from the n. commissuralis .