These experiments further define the organization of peptidergic pathways in the paravertebral sympathetic system of the bullfrog. Populations of axons and synaptic boutons in sympathetic ganglia 9 and 10 were found to express calcitonin gene-related peptide-like immunoreactivity (CGRP-IR) and substance P-like immunoreactivity (SP-IR). CGRP-IR is present in fibers that run through the ganglia and in boutons that make contact with almost half of the principal neurons. SP-IR is also present in fibers within the ganglia and in a rare class of synaptic boutons that are found on less than 1% of the principal neurons. Both forms of immunoreactivity are coexpressed in some nerve fibers and in the rare synaptic boutons that contain SP-IR. Neuropeptide Y-like immunoreactivity (NPY-IR), a marker for C-type postganglionic neurons, was used to identify the postsynaptic targets of boutons containing CGRP-IR and SP-IR. Ninety-five percent of the ganglion cells contacted by CGRP-IR boutons were negative for NPY-IR and are therefore likely to be B-type postganglionic neurons. Similarly, 100% of the ganglion cells contacted by boutons containing SP-IR were negative for NPY-IR. Lesions of the sympathetic chain demonstrated that synaptic boutons containing CGRP-IR arise from neurons whose axons enter the chain rostral to ganglion 7. Cutting the chain between ganglia 8 and 9 eliminates all preganglionic B and C inputs to ganglia 9 and 10. The destruction of the preganglionic C pathway by this lesion was verified by staining ganglia 9 and 10 for luteinizing hormone releasing hormone (LHRH). This lesion also eliminated boutons containing CGRP-IR and drastically reduced the number of ganglionic fibers that stained for CGRP-IR and SP-IR. By contrast, cutting the sympathetic chain between ganglia 6 and 7 spared LHRH-IR in the preganglionic C pathway but still eliminated the boutons that normally express CGRP-IR and reduced the amount of staining for SP-IR. CGRP-IR in the sympathetic ganglia arises from preganglionic and sensory neurons whereas ganglionic SP-IR is purely sensory in origin. In the spinal cord, the preganglionic B and C neurons that innervate ganglia 9 and 10 are located in different segments. In segments that contain preganglionic B cells, but not those that contain C cells, there were 243 +/- 37 (mean +/- SD) neurons in the intermediolateral cell column that express CGRP-IR. However, no cell bodies containing SP-IR were found in this region of the spinal cord.(ABSTRACT TRUNCATED AT 400 WORDS)