This study investigated possible sites of contact of nerve fibers containing a range of putative neurotransmitter substances onto neurons in the cat ventral medulla oblongata concerned with autonomic, particularly cardiovascular, regulation. The parasympathetic preganglionic neurons of the nucleus ambiguous (correction of ambiguus) were identified by retrograde horseradish peroxidase tracing from the vagus nerve, and the groups of neurons in the A1 and C1 cell areas and the raphe nucleus by catecholamine enzyme or 5-hydroxytryptamine (5-HT) immunohistochemistry, respectively. Immunoreactive (-ir)nerve fibers and terminals in the vicinity if these neurons were visualized by subjecting the sections to a dual-staining technique using a brown peroxidase-diaminobenzidine reaction product and a blue alkaline phosphatase-Fast blue reaction product. By employing monochrome photography with combinations of blue and orange-red filters, it was possible to discriminate neural elements displaying one or the other reaction product, or colocalization of reaction products. The results revealed the presence of calcitonin gene-related peptide (CGRP) and galanin (GAL)-ir in some motoneurons of the nucleus ambiguus, but not in those innervating the heart via the cardiac vagus nerve. The latter group of parasympathetic efferent neurons were found to be densely innervated by fibers immunoreactive for dopamine beta-hydroxylase (DBH, indicating noradrenaline), glycine (GLY), gamma-aminobutyric acid (GABA), 5-HT, enkephalin (ENK), neuropeptide Y (NPY), substance P (SP), and thyrotropin-releasing hormone (TRH), and, to a lesser extent, by other neuropeptide-ir fibers. The catecholamine cells of the rostral C1 and caudal A1 groups showed a broadly similar pattern of innervation, most noticeably by fibers immunoreactive for DBH, GABA, 5-HT, cholecystokinin (CCK), CGRP, ENK, GAL, NPY, and SP. The 5-HT-ir neurons of the raphe nucleus, some also containing SP, TRH, ENK, or corticotropin-releasing factor (CRF)-ir, were most prominently innervated by terminals containing DBH, GABA, CCK, ENK, NPY, TRH, somatostatin (SRIF), and vasoactive intestinal polypeptide (VIP)-ir. Although the proof that these groups of neurons receive functional synaptic contacts from the immunoreactive fibers awaits further ultrastructural studies, the results do suggest that a wide range of putative transmitters may influence the activity of efferent neurons in the cat medulla controlling autonomic functions.