A combination of immunocytochemical and electron microscopic methods were employed to assess the organization of the trigeminal (V) spinal tract in adult rats. Immunostaining was employed at the light microscopic level to selectively label large myelinated (by using antibodies against neurofilament protein) and small unmyelinated (by using antibodies against calcitonin gene-related peptide) primary afferents. In addition, the plant lectin Bandeiraea simplicifolia-I was employed to histochemically label small unmyelinated primary afferents. Results from these experiments indicated that larger myelinated axons were distributed throughout the cross-sectional extent of the V spinal tract (TrV), whereas smaller fibers were most numerous just below the pial surface. These results were confirmed with quantitative electron microscopy which demonstrated that the central portion of the V sensory root and TrV were composed primarily of larger myelinated fibers, whereas the periphery of the root and the portion of TrV just below the pial surface contained a higher percentage of smaller myelinated and unmyelinated axons. When considered together with results regarding the birthdates of neurochemically defined classes of V ganglion cells (White et al. [1994] J. Comp. Neurol. 350:397-411), these results suggest that TrV is laid down in a chronotopic fashion with the first axons forming its deeper portion and later arriving axons being added more superficially.