An antiserum to alpha-melanocyte-stimulating hormone (alpha-MSH) was found to contain antibodies to at least two types of determinants on the alpha-MSH peptide: one is present only on the free peptide, the other is shared with neurofilaments. Immunoblots from mouse brain showed the neurofilament crossreactivity to be located on proteins in the Mr 140,000 range. The neurofilament-crossreactive portion of the antiserum could be selectively absorbed out with a cytoskeletal preparation, which abolished all affinity of the antiserum to the retina but did not affect the labeling pattern in the pituitary. Absorptions with desacetyl-alpha-MSH and corticotropin seemed to indicate that the determinant shared with neurofilaments is not located at either end of the alpha-MSH peptide, but somewhere in between. The immunohistochemical labeling of the retina with the alpha-MSH antiserum was compared to the labeling with monoclonal antibodies against Mr 200,000 neurofilaments. In the adult retina the alpha-MSH-like immunoreactivity was found to be slightly more widespread; most consistently it was detectable in cell bodies of large ganglion cells, whereas the heavy neurofilament subunit was absent from somata and proximal axons of these cells. In the developing mouse brain, expression of the heavy subunit was found to lag 2-3 wk behind expression of the Mr 140,000 proteins. This confirms previous reports of a more restricted distribution and late expression of high molecular weight neurofilaments as compared to the lower subunits.