To further address the hypothesis that cholecystokinin (CCK) in the medullary dorsal horn (MDH) arises from intrinsic or higher‐order neurons. CCK‐8‐specific radioimmunoassay (RIA) and immunohistochemichal (IHC) experiments were carried out in adult rats after trigeminal tractotomy. RIA of punches from deafferented superficial layers of the MDH revealed no significant change in CCK levels vs. the control right side. In this same area, IHC revealed modest reductions in CCK, gastrin, and substance P staining. Calcitonin gene‐related peptide (CGRP) staining was reduced substantially. Gastrin immunoreactive cell bodies, present normally in inner lamina II, were reduced in number. RIA and IHC methods were also used to assess MDH CCK concentrations in adult rats subjected to left infraorbital nerve section at birth. The left medulla contained significantly higher levels of CCK than the control right medulla (1.27 ± 0.19 vs. 0.97 ± 0.11 ng/mg protein). IHC revealed a dense band of CCK‐like staining in laminae I and II ipsi‐ and contralateral to the lesion. Thus, neonatal deafferentation elevates medullary CCK. To determine if the neonatal lesion‐induced increase in medullary CCK is due to primary afferent or higher‐order reorganization, RIA and IHC experiments were run after infraorbital nerve section at birth and trigeminal tractotomy in adulthood. RIA revealed no significant change in CCK levels caudal to the tractotomy, although they were higher than control levels in 9 of 12 cases. IHC revealed modest reductions in CCK, substance P, and gastrin staining that resembled the reductions observed in tractotomy‐alone cases. These data suggest that (1) most MDH CCK is of non‐primary afferent origin, (2) gastrin immunoreactivity in layer II probably originates in CCK‐containing cells intrinsic to layer II, the expression of which is dependent upon trigeminal primary afferent input, (3) neonatal V deafferentation induces increased CCK in the superficial MDH, oflecting reorganized intrinsic or higher‐order inputs, and (4) higher‐order substance P in the MDH is robust. © 1992 Wiley‐Liss, Inc.