Endopeptidase-24.11 is a widely distributed cell surface enzyme with a role in inactivating some neuropeptides and peptide hormones. In the central nervous system it has been implicated in the metabolism of enkephalins and tachykinins, neuropeptides which are expressed by neurons projecting to the substantia nigra. Two immunochemical methods have been used to reveal the ultrastructural localization of endopeptidase-24.11 and substance P in the substantia nigra of piglets. In the first approach, substance P was revealed by immunoperoxidase staining using the rat monoclonal antibody, NC1, and endopeptidase-24.11 by 1 nm colloidal gold using an affinity-purified rabbit polyclonal antibody, both being applied at the pre-embedding stage. NC1 was shown to be highly specific for substance P, with negligible cross-reactivity with neurokinins A and B. The specificity of the immunostaining was confirmed by processing all sections for both markers, even when only one primary antibody was applied. In the second approach, ultrathin cryosections were immunostained using gold particles of different diameters. In a survey of electron micrographs, 80% of the silver-enhanced gold particles were touching neuronal membranes, consistent with the known topology of endopeptidase-24.11. Endopeptidase-24.11 immunoreactivity was observed both on membranes of axons and on pre- and postsynaptic elements. Substance P immunoreactivity was seen within some boutons, apparently associated with vesicles, and in axons. In doubly stained sections, many examples of immunopositive gold-labelled boutons (i.e. endopeptidase-24.11-immunoreactive-positive) containing immunoperoxidase reaction product (i.e. substance P-immunoreactive-positive) were recorded. In ultrathin cryosections, substance P immunoreactivity was mainly observed in dense-core vesicles within boutons, some of which also showed membrane-associated gold particles marking endopeptidase-24.11 immunoreactivity. This is the first demonstration of endopeptidase-24.11 by immunogold at the electron microscopic level and the first demonstration of the ultrastructural co-localization of a membrane peptidase and its putative neuropeptide target. The results lend strong support to the view that endopeptidase-24.11 has a physiological role in the metabolism of substance P, but do not exclude a role in the inactivation of other neuropeptides.