We previously reported the abnormal expression of hindlimb receptive fields in the stump representation of the primary somatosensory cortex (SI) in rats that sustained neonatal forelimb removal when cortical gamma-aminobutyric acid (GABA) receptors were pharmacologically blocked (Lane et al. [1997] J. Neurophysiol. 77:2723-2735). In this study, we attempted to identify the substrate for this functional modification. Three potential substrates were examined: 1) changes in intracortical connections within SI; 2) alterations in the projection pattern of thalamocortical afferents from the ventroposterior lateral (VPL) nucleus to SI; and 3) changes in the receptive fields of thalamocortical neurons. We used biotinylated dextran amine and Phaseolus vulgaris leucoagglutinin to examine the intracortical projections associated with the stump and hindlimb representations of SI. True Blue and Diamidino Yellow were used to study the organization of the VPL projections to SI. Finally, single-unit recordings from VPL neurons were made to examine the functional organization of this nucleus in neonatally amputated adult rats. Tracer studies demonstrated no significant change in the intracortical connections or VPL projections associated with the stump and hindlimb SI in neonatally amputated rats. Recordings from VPL of neonatally manipulated rats revealed a small, but significant, population of cells (19.0%) within the stump representation that had dual stump and hindlimb receptive fields. Thus, the data suggest that the functional reorganization observed in SI of neonatally amputated rats may reflect functional alterations occurring in its thalamic inputs.