The method of retrograde axonal transport of horseradish peroxidase (HRP) was used to identify the locations of cells of origin of the spinothalamic tract in the cat. Injections of from 0.2-3.0 microliter of 30% HRP were made unilaterally in various regions of the somatosensory thalamus. Massive injections of the caudal thalamus in several cats showed the spinothalamic cells of origin to be located mainly in laminae I, VII and VIII in the lumbar enlargement, and in laminae I, V and VII-VIII in the cervical enlargement. Small injections of HRP were made into the three major spinothalamic terminal zones in the thalamus, to determine the laminar origins(s) of the spinal projections to each zone. Neurons in lamina I in both cervical and lumbar enlargements were found to project almost exclusively to the rostral VB-caudal VL border zone. A small number of neurons in laminae VII and VIII also project there but a larger number project to the intralaminar region. Neurons projecting to the PO regions were located mainly in laminae IV and V. This anatomical segregation of thalamic afferents probably reflects a functional segregation of input, since the functional properties of spinal neurons vary according to their laminar location. Comparison of these data with the differential projection of spinothalamic neurons in the rat and monkey indicate that it is unlikely that the proposed "paleo-" and "neospinothalamic" systems would arise from anatomically separate groups of spinal neurons.