Pancreatic ganglia are formed by neural crest-derived precursors, are innervated by enteric neurons, and contain neuropeptides. In addition, the enzyme NADPH-diaphorase is located in a subset of enteric and pancreatic neurons. The expression of neural markers (GAP-43 and NC-1), neurotransmitter-related markers (including neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), gastrin-releasing peptide (GRP), galanin (GAL), dopamine beta hydroxylase (DBH), substance P (SP), calcitonin gene-related peptide (CGRP)), and NADPH-diaphorase was studied in the fetal and neonatal rat gut and pancreas (E12-P28) in situ and in vitro. NC-1, GAP-43 and DBH-immunoreactive cells were found in the primordial stomach on day E12, and in the pancreas on day E13, along with NPY in endocrine cells. Pancreatic NPY-immunoreactive neurons were detected by day E18. CGRP was seen in the foregut at day E12 but not in the pancreas until day E14. Other neuropeptides (SP, GAL, GRP and VIP) all appeared in the foregut earlier than in the pancreas. NADPH-diaphorase activity was first found in situ in foregut neurons on day E13, and in the pancreas on day E14, but seen in explants a day earlier. These observations show that development of neurons occurs earlier in the gut than in the pancreas, and that NADPH-diaphorase activity appears earlier than the immunoreactivities of the neuropeptides.