During development, the earliest generated neurons of the mammalian telencephalon reside in a region of the white matter, the subplate, just beneath the cortical plate. Neurons in the subplate are only transiently present in the telencephalon: shortly after birth in the cat the majority have disappeared. During their brief life, however, subplate neurons mature; they extend long-distance and local projections, and express immunoreactivity for GABA and several neuropeptides. In the present study we examined the relation between possible transmitter phenotypes of subplate neurons and their connectivity. To do so, we used a double-label technique in which immunohistochemistry for neuropeptide Y (NPY), somatostatin (SRIF) or calbindin (CaBP) was combined with retrograde tracing. Experiments were performed in neonatal cats and in ferret kits at equivalent postconceptional ages, times when subplate neurons are numerous. Subplate neurons immunoreactive for neuropeptides and CaBP could be double-labelled by an injection of retrograde tracer either into the cortical plate or the white matter, indicating that this particular subset of subplate neurons can make local circuit projections. In contrast, peptide or CaBP immunoreactive subplate neurons could never be retrogradely labelled from a tracer injection into the thalamus. Taken together, these observations indicate that subplate neurons immunoreactive for NPY, SRIF and CaBP are likely to be interneurons exclusively. On the other hand, subplate neurons with long-distance projections to the thalamus or the contralateral hemisphere could be labelled by the retrograde transport of D-[3H]aspartate, suggesting that at least some projection subplate neurons might use an excitatory amino acid as a neurotransmitter. These results indicate that there is a defined relationship between the putative transmitter phenotypes of subplate neurons and their patterns of projection. Interneurons of the subplate express peptidergic properties while projection neurons to the thalamus may use an excitatory amino acid. Thus, these basic organizational features of the transient subplate are reminiscent of those found in the adult cortical layers.