A vesicular glutamate transporter, VGluT2, has been suggested to be the transporter utilized in the thalamocortical pathway. We examined the reliability of this marker in identifying and discriminating thalamic terminals in adult and developing ferret visual cortex. We studied brain sections stained for the transporter protein and/or anterogradely filled thalamocortical or intracortical axons, by using light, confocal, and electron microscopy. Under light microscopy, VGluT2 immunoreactivity (ir) in adult animals [past postnatal day (P)90] and in neonatal animals as early as P27 formed a dense band in layer 4 and appeared as scattered puncta in layers 6 and 1. Confocal dual‐labeling analyses of P46 and adult striate cortices indicated that VGluT2 was present in thalamocortical axons, suggesting that thalamic projections utilize this transporter during postnatal development as well as adulthood. In contrast, extracellularly filled intracortical axons failed to colocalize with VGluT2‐ir, suggesting that no significant terminal population originating in cortex contained VGluT2 in layer 4. Electron microscopic analysis revealed that, in adult layer 4, VGluT2‐ir was present in large terminals, forming asymmetric synapses. Similar to anterogradely labeled thalamocortical terminals, VGluT2‐ir synaptic terminals were different from their unlabeled counterparts in terms of terminal area (0.6 vs. 0.3 μm), synaptic length (486 vs. 353 nm), and preference for synapsing on spines (77% vs. 59%). Moreover, no significant differences were found between VGluT2‐ir and anterogradely labeled thalamocortical terminals. Comparable similarities were also demonstrated at P46. These results indicate that thalamocortical terminals in layer 4 of visual cortex utilize VGluT2 and suggest that this marker can be used to identify thalamic axons specifically in adult and developing animals. J. Comp. Neurol. 484:458–473, 2005. © 2005 Wiley‐Liss, Inc.